Effect of macrophage-to-myofibroblast transition on silicosis.

BACKGROUND: The aim was to explore the effect of macrophage polarization and macrophage-to-myofibroblast transition (MMT) in silicosis. METHODS: Male Wistar rats were divided into a control group and a silicosis group developed using a HOPE MED 8050 dynamic automatic dusting system. Murine macrophage MH-S cells were randomly divided into a control group and an SiO2 group. The pathological changes in lung tissue were observed using hematoxylin and eosin (HE) and Van Gieson (VG) staining. The distribution and location of macrophage marker (F4/80), M1 macrophage marker (iNOS), M2 macrophage marker (CD206), and myofibroblast marker (α-smooth muscle actin [α-SMA]) were detected using immunohistochemical and immunofluorescent staining. The expression changes in iNOS, Arg, α-SMA, vimentin, and type I collagen (Col I) were measured using Western blot. RESULTS: The results of HE and VG staining showed obvious silicon nodule formation and the distribution of thick collagen fibers in the lung tissue of the silicosis group. Macrophage marker F4/80 increased gradually from 8 to 32 weeks after exposure to silica. Immunohistochemical and immunofluorescent staining results revealed that there were more iNOS-positive cells and some CD206-positive cells in the lung tissue of the silicosis group at 8 weeks. More CD206-positive cells were found in the silicon nodules of the lung tissues in the silicosis group at 32 weeks. Western blot analysis showed that the expressions of Inducible nitric oxide synthase and Arg protein in the lung tissues of the silicosis group were upregulated compared with those of the control group. The results of immunofluorescence staining showed the co-expression of F4/80, α-SMA, and Col I, and CD206 and α-SMA were co-expressed in the lung tissue of the silicosis group. The extracted rat alveolar lavage fluid revealed F4/80+α-SMA+, CD206+α-SMA+, and F4/80+α-SMA+Col I+ cells using immunofluorescence staining. Similar results were also found in MH-S cells induced by SiO2. CONCLUSIONS: The development of silicosis is accompanied by macrophage polarization and MMT.

Paeoniflorin mitigates MMP-12 inflammation in silicosis via Yang-Yin-Qing-Fei Decoction in murine models.

BACKGROUND: Silicosis presents a significant clinical challenges and economic burdens, with Traditional Chinese Medicine (TCM) emerging as a potential therapeutic avenue. However, the precise effects and mechanisms of TCM in treating silicosis remain uncertain and subject to debate. OBJECTIVE: The study aims to elucidate the therapeutic role and mechanisms of the Yang-Yin-Qing-Fei Decoction (YYQFD) and its key component, paeoniflorin, in silicosis using a murine model. METHODS: Silicotic mice were treated with YYQFD, pirfenidone (PFD), or paeoniflorin. RAW264.7 cells and mouse lung fibroblasts (MLF) were stimulated with silica, matrix metalloproteinase-12 (MMP-12), or TGF-ß1, followed by treatment with paeoniflorin, PFD, or relevant inhibitors. YYQFD constituents were characterized using High-Performance Liquid Chromatography (HPLC). Lung fibrosis severity was assessed via histopathological examination, micro-CT imaging, lung functions, and Western blot analysis. Transcriptome sequencing and bioinformatics analysis were employed to delineate the gene expression profile and target genes modulated by YYQFD in silicosis. RESULTS: Treatment with YYQFD ameliorated silica-induced lung fibrosis. Transcriptome sequencing identified MMP-12 as a potential common target of YYQFD and PFD. Additionally, a potential pro-inflammatory role of MMP-12, regulated by silica-induced TLR4 signaling pathways, was revealed. Paeoniflorin, one of the most distinctive compounds in YYQFD, attenuated silica-induced MMP-12 increase and its derived inflammatory factors in macrophages through a direct binding effect. Notably, paeoniflorin treatment exerted anti-fibrotic effects by inhibiting MMP-12-derived inflammatory factors and TGF-ß1-induced myofibroblast differentiation in silica-exposed mice. CONCLUSIONS: This study underscores paeoniflorin as one of the most principal bioactive compounds in YYQFD, highlighting its capacity to attenuate lung inflammation driven by macrophage-derived MMP-12 and reduce lung fibrosis both in vivo and in vitro.

ISRIB inhibits the senescence of type II pulmonary epithelial cells to alleviate pulmonary fibrosis induced by silica in mice.

The role and mechanisms of integrated stress response inhibitor (ISRIB) on silicosis are still not well defined. In the present study, the effects of ISRIB on cellular senescence and pulmonary fibrosis in silicosis were evaluated by RNA sequencing, micro-computed tomography, pulmonary function assessment, histological examination, and Western blot analysis. The results showed that ISRIB significantly reduced the degree of pulmonary fibrosis in mice with silicosis and reduced the expression of type I collagen, fibronectin, α-smooth muscle actin, and transforming growth factor-ß1. Both in vivo and in vitro results showed that ISRIB reversed the expression of senescence-related factors ß-galactosidase, phosphor-ataxia telangiectasia mutated, phosphor-ataxia telangiectasia and Rad3-related protein, p-p53, p21, p16, and plasminogen activator inhibitor type 1. The aforementioned results were consistent with the sequencing results. These findings implied that ISRIB might reduce the degree of pulmonary fibrosis in mice with silicosis by inhibiting the cellular senescence of alveolar epithelial cell type II.

Ac-SDKP promotes KIF3A-mediated ß-catenin suppression through a ciliary mechanism to constrain silica-induced epithelial-myofibroblast transition.

Kinesin family member 3 A (KIF3A) decrease have been reported in silicotic patients and rats. However, the detailed mechanisms of KIF3A in silicosis remain unknown. In this study, we demonstrated that KIF3A effectively blocked the expression of ß-catenin and downstream myocardin-related transcription factor (MRTF)-A/serum response factor (SRF) signaling, thus inhibiting silica-induced epithelial-myofibroblast transition (EMyT). Moreover, KIF3A was identified as a downstream mediator of an antifibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Knockdown of KIF3A expression reactivated ß-catenin/myocardin-related transcription factor (MRTF)-A/serum response factor (SRF) signaling that was attenuated by Ac-SDKP in vitro. Collectively, our findings suggest that Ac-SDKP plays its anti-fibrosis role via KIF3A-mediated ß-catenin suppression, at least in part, in both in vivo model of silicosis and in vitro model of EMyT.

Exercise training inhibits macrophage-derived IL-17A-CXCL5-CXCR2 inflammatory axis to attenuate pulmonary fibrosis in mice exposed to silica.

Exposure to crystalline silica leads to health effects beyond occupational silicosis. Exercise training's potential benefits on pulmonary diseases yield inconsistent outcomes. In this study, we utilized experimental silicotic mice subjected to exercise training and pharmacological interventions, including interleukin-17A (IL-17A) neutralizing antibody or clodronate liposome for macrophage depletion. Findings reveal exercise training's ability to mitigate silicosis progression in mice by suppressing scavenger receptor B (SRB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and Toll-like receptor 4 (TLR4) pathways. Macrophage-derived IL-17A emerges as primary source and trigger for silica-induced pulmonary inflammation and fibrosis. Exercise training effectively inhibits IL-17A-CXC motif chemokine ligand 5 (CXCL5)-Chemokine (C-X-C motif) Receptor 2 (CXCR2) axis in silicotic mice. Our study evidences exercise training's potential to reduce collagen deposition, preserve elastic fibers, slow pulmonary fibrosis advancement, and enhance pulmonary function post silica exposure by impeding macrophage-derived IL-17A-CXCL5-CXCR2 axis.

Quercetin Alleviates Pulmonary Fibrosis in Silicotic Mice by Inhibiting Macrophage Transition and TGF-ß-Smad2/3 Pathway.

Silicosis is a pulmonary disease caused by the inhalation of silica. There is a lack of early and effective prevention, diagnosis, and treatment methods, and addressing silicotic fibrosis is crucial. Quercetin, a flavonoid with anti-carcinogenic, anti-inflammatory, and antiviral properties, is known to have a suppressive effect on fibrosis. The present study aimed to determine the therapeutic effect of quercetin on silicotic mice and macrophage polarity. We found that quercetin suppressed silicosis in mice. It was observed that SiO2 activated macrophage polarity and the macrophage-to-myofibroblast transition (MMT) by transforming the growth factor-ß (TGF-ß)-Smad2/3 signaling pathway in silicotic mice and MH-S cells. Quercetin also attenuated the MMT and the TGF-ß-Smad2/3 signaling pathway in vivo and in vitro. The present study demonstrated that quercetin is a potential therapeutic agent for silicosis, which acts by regulating macrophage polarity and the MMT through the TGF-ß-Smad2/3 signaling pathway.

Effect of Sex Differences in Silicotic Mice.

Mechanisms of silicosis, caused by the inhalation of silica are still unclear, and the effect of sex on silicosis has rarely been reported. The purpose of this study was to investigate whether sex affects the silicotic lesions and the progressive fibrotic responses in silicosis. Our study showed that sex had no significant effect on the area of silicon nodules and the collagen deposition after a one-time bronchial perfusion of silica. Immunohistochemical staining showed that CD68 and the transforming growth factor-ß1 (TGF-ß1) were positive in male and female silicotic mice. In addition, the western blot results showed that the fibrosis-related factors type I collagen (COL I), α-smooth muscle actin (α-SMA), vimentin, TGF-ß1, p-SMAD2/3, inflammatory-related factors interleukin 6 (IL 6), interleukin 1ß (IL 1ß), and senescence-related factors p16 and p21 were up-regulated in silicotic mice and there was no difference between female or male mice exposed to silica. The expression of TGF-ß1, p-SMAD2/3, p16, and p21 were downregulated in the early stage of female silicotic mice, compared to the males. Thus, despite differences in the expression of certain factors, there was no overall difference in the progressive fibrosis between female and male mice in silicosis. These results thus provide a new perspective for studying the pathological development of silicosis.

Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica.

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent models continue to be an essential tool for studying human silicosis. For silicosis, the classic pathological process needs to be inducible via the inhalation of freshly generated quartz particles, which means specifically inducing human occupational disease. This study described a technique to establish a silicosis rat model with inhalation of silica via the whole body in an inhalation chamber, which is simple, easy to operate, and effectively mimics the pathological dynamic evolution process of silicosis. Further, the technique had good repeatability with no surgery involved. The inhalation exposure system was fabricated, validated, and used for toxicology studies on respirable particle inhalation. The critical components were as follows: (1) bulk dry SiO2 powder generator adjusted with an air-flow controller; (2) 0.3 m3 whole-body inhalation exposure chamber accommodating up to 20 adult rats; (3) a monitoring and control system for regulating oxygen concentration, temperature, humidity, and pressure in real-time; and (4) a barrier and waste disposal system for protecting laboratory technicians and the environment. In summary, the present protocol reports the inhalation via the whole body, and the inhalation chamber created a reliable, reasonable, and repeatable rat silicotic model with low mortality, less injury, and more protection.

Quercetin Alleviates Pulmonary Fibrosis in Mice Exposed to Silica by Inhibiting Macrophage Senescence.

Quercetin exerts anti-inflammatory, anti-oxidant and other protective effects. Previous studies have shown that senescent cells, such as fibroblasts and type II airway epithelial cells, are strongly implicated in the development of pulmonary fibrosis pathology. However, the role of senescent macrophages during silicosis remains unclear. We investigated the effects of quercetin on macrophage senescence and pulmonary fibrosis, and explored underlying mechanisms. Mice were randomized to six model groups. Vitro model was also established by culturing RAW264.7 macrophages with silica (SiO2). We examined the effects of quercetin on fibrosis, senescence-associated ß-galactosidase (SA-ß-Gal) activity, and senescence-specific genes (p16, p21, and p53). We showed that quercetin reduced pulmonary fibrosis and inhibited extracellular matrix (ECM) formation. Quercetin also attenuated macrophage senescence induced by SiO2 both in vitro and in vivo. In addition, quercetin significantly decreased the expressions of the senescence-associated secretory phenotype (SASP), including proinflammatory factors (interleukin-1α (Il-1α), Il-6, tumor necrosis factor-α (TNF-α), and transforming growth factor-ß1 (TGF-ß1)) and matrix metalloproteinases (MMP2, MMP9, and MMP12). In conclusion, quercetin mediated its anti-fibrotic effects by inhibiting macrophage senescence, possibly via SASP.

Minute Cellular Nodules as Early Lesions in Rats with Silica Exposure via Inhalation.

Mechanisms of silicosis have yet to be clarified, and pathological conditions are inaccurately described in some experimental studies on silicosis. This study was aimed at describing initial lesions in silicosis, as observed in rats with silica exposure via inhalation, and major histopathologic alterations. Male Wistar rats were exposed to silica for 24 weeks. Hematoxylin and eosin staining indicated the presence of "cellular nodule+ macrophage alveolitis" in rats exposed to silica from the 2-16 weeks time points and "fibrotic cellular + cellular nodule" in rats exposed to silica via inhalation for 24 weeks. By immunohistochemistry, the following were noted: a continual increase in the positive expression of CD68 in macrophages in the lungs of rats exposed to silica; hyperplasia in alveolar type II cells (AT2); loss of original phenotypes in fibrotic cellular nodules, macrophages, and AT2 cells; loss of endothelial cells in silicotic nodules; and positive expression of α-smooth muscle actin in macrophages. Typical pathological changes in silicosis were also summarized. Among these changes were macrophage alveolitis, cellular nodules, and fibrotic cellular nodules, including an increase in minute cellular nodules in the early stages and the formation of fibrotic cellular nodules in the late stages.

Thalidomide Alleviates Pulmonary Fibrosis Induced by Silica in Mice by Inhibiting ER Stress and the TLR4-NF-κB Pathway.

Silicosis is the most prevalent occupational disease in China. It is a form of pulmonary fibrosis caused by the inhalation of silicon particles. As there is no cure for the potentially lethal and progressive condition, the treatment of silicotic fibrosis is an important and difficult problem to address. Thalidomide, a drug with anti-inflammatory and immunoregulatory properties, has been reported to have lung-protective effects. The purpose of this study was to observe the therapeutic effect of thalidomide on silicotic mice and to determine the protective mechanism. By using silicotic mice models and MH-S cells, we found the expression of endoplasmic reticulum stress (ER stress) and Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) pathway as well as inflammation-related factors were upregulated in the macrophages of silicotic mice. The same indexes were detected in silica-stimulated MH-S cells, and the results were consistent with those in vivo. That is, silica activated ER stress and the TLR4-NF-κB pathway as well as the inflammatory response in vitro. Treating both silicotic mice and silica-stimulated MH-S cells with thalidomide inhibited ER stress and the TLR4-NF-κB pathway as well as the inflammatory response. The present study demonstrates thalidomide as a potential therapeutic agent against silicosis.

Oxamate Attenuates Glycolysis and ER Stress in Silicotic Mice.

Glycolysis and ER stress have been considered important drivers of pulmonary fibrosis. However, it is not clear whether glycolysis and ER stress are interconnected and if those interconnections regulate the development of pulmonary fibrosis. Our previous studies found that the expression of LDHA, a key enzyme involved in glycolysis, was increased in silica-induced macrophages and silicotic models, and it was closely related to silicosis fibrosis by participating in inflammatory response. However, whether pharmacological inhibition of LDHA is beneficial to the amelioration of silicosis fibrosis remains unclear. In this study, we investigated the effects of oxamate, a potent inhibitor of LDHA, on the regulation of glycolysis and ER stress in alveolar macrophages and silicotic mice. We found that silica induced the upregulation of glycolysis and the expression of key enzymes directly involved in ER stress in NR8383 macrophages. However, treatment of the macrophages and silicotic mice with oxamate attenuated glycolysis and ER stress by inhibiting LDHA, causing a decrease in the production of lactate. Therefore, oxamate demonstrated an anti-fibrotic role by reducing glycolysis and ER stress in silicotic mice.

MicroRNA-411-3p inhibits bleomycin-induced skin fibrosis by regulating transforming growth factor-ß/Smad ubiquitin regulatory factor-2 signalling.

Skin fibrosis, which is characterized by fibroblast proliferation and increased extracellular matrix, has no effective treatment. An increasing number of studies have shown that microRNAs (miRNAs/miRs) participate in the mechanism of skin fibrosis, such as in limited cutaneous systemic sclerosis and pathological scarring. The objective of the present study was to determine the role of miR-411-3p in bleomycin (BLM)-induced skin fibrosis and skin fibroblast transformation. Using Western blot analysis and real-time quantitative polymerase chain reaction assess the expression levels of miR-411-3p, collagen (COLI) and transforming growth factor (TGF)-ß/Smad ubiquitin regulatory factor (Smurf)-2/Smad signalling factors both in vitro and in vivo with or without BLM. To explore the regulatory relationship between miR-411-3p and Smurf2, we used the luciferase reporter assay. Furthermore, miR-411-3p overexpression was identified in vitro and in vivo via transfection with Lipofectamine 2000 reagent and injection. Finally, we tested the dermal layer of the skin using haematoxylin and eosin and Van Gieson's staining. We found that miR-411-3p expression was decreased in bleomycin (BLM)-induced skin fibrosis and fibroblasts. However, BLM accelerated transforming growth factor (TGF)-ß signalling and collagen production. Overexpression of miR-411-3p inhibited the expression of collagen, F-actin and the TGF-ß/Smad signalling pathway factors in BLM-induced skin fibrosis and fibroblasts. In addition, miR-411-3p inhibited the target Smad ubiquitin regulatory factor (Smurf)-2. Furthermore, Smurf2 was silenced, which attenuated the expression of collagen via suppression of the TGF-ß/Smad signalling pathway. We demonstrated that miR-411-3p exerts antifibrotic effects by inhibiting the TGF-ß/Smad signalling pathway via targeting of Smurf2 in skin fibrosis.

Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment.

Glycolytic reprogramming is an important metabolic feature in the development of pulmonary fibrosis. However, the specific mechanism of glycolysis in silicosis is still not clear. In this study, silicotic models and silica-induced macrophage were used to elucidate the mechanism of glycolysis induced by silica. Expression levels of the key enzymes in glycolysis and macrophage activation indicators were analyzed by Western blot, qRT-PCR, IHC, and IF analyses, and by using a lactate assay kit. We found that silica promotes the expression of the key glycolysis enzymes HK2, PKM2, LDHA, and macrophage activation factors iNOS, TNF-α, Arg-1, IL-10, and MCP1 in silicotic rats and silica-induced NR8383 macrophages. The enhancement of glycolysis and macrophage activation induced by silica was reduced by Ac-SDKP or siRNA-Ldha treatment. This study suggests that Ac-SDKP treatment can inhibit glycolytic reprogramming in silica-induced lung macrophages and silicosis.

OC-STAMP Overexpression Drives Lung Alveolar Epithelial Cell Type II Senescence in Silicosis.

Cellular senescence has been considered an important driver of many chronic lung diseases. However, the specific mechanism of cellular senescence in silicosis is still unknown. In the present study, silicotic rats and osteoclast stimulatory transmembrane protein (Ocstamp) overexpression of MLE-12 cells were used to explore the mechanism of OC-STAMP in cellular senescence in alveolar epithelial cell type II (AEC2). We found an increasing level of OC-STAMP in AEC2 of silicotic rats. Overexpression of Ocstamp in MLE-12 cells promoted epithelial-mesenchymal transition (EMT), endoplasmic reticulum (ER) stress, and cellular senescence. Myosin heavy chain 9 (MYH9) was a potential interacting protein of OC-STAMP. Knockdown of Ocstamp or Myh9 inhibited cellular senescence in MLE-12 cells transfected with pcmv6-Ocstamp. Treatment with 4-phenylbutyrate (4-PBA) to inhibit ER stress also attenuated cellular senescence in vitro or in vivo. In conclusion, OC-STAMP promotes cellular senescence in AEC2 in silicosis.

Ac-SDKP Attenuates Activation of Lung Macrophages and Bone Osteoclasts in Rats Exposed to Silica by Inhibition of TLR4 and RANKL Signaling Pathways.

BACKGROUND: Silica-induced inflammatory activation is associated with silicosis and various non-respiratory conditions. The present study was designed to examine the anti-inflammatory effects of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on lung macrophages and bone osteoclasts after silica inhalation in rats. METHODS: Wistar rats and NR8383 and RAW 264.7 cell lines were used in the present study. The receptor activator of nuclear factor kappa-B ligand (RANKL) and toll-like receptor 4 (TLR4) signaling pathways was measured in the lung tissue of rats or NR8383/RAW 264.7 cells exposed to silica. The microarchitecture of the trabecular bone in the tibia and femur was evaluated in silicotic rats. Furthermore, the roles of Ac-SDKP on silicotic rats, silica-treated NR8383/RAW 264.7 cells, and RANKL-induced osteoclast differentiation were studied. RESULTS: The data indicated that silica inhalation might activate the RANKL and TLR4 signaling pathways in lung macrophages, thus inducing the lung inflammatory and proteolytic phenotype of macrophages and osteoclasts in lung and bone. Ac-SDKP maintained the lung elastin level by inhibiting lung inflammation and macrophage activation via the RANKL and TLR4 signaling pathways. Ac-SDKP also attenuated the reduction in femoral bone mineral density in silicotic rats by inhibiting osteoclast differentiation via the RANKL signaling pathway. CONCLUSION: Our findings support the hypothesis that inhalation of crystalline silica induces activation of lung macrophages and bone osteoclasts via the RANKL and TLR4 signaling pathways. Ac-SDKP has the potential to stabilize lung homeostasis and bone metabolism.

Transcriptomic analysis identifies upregulation of secreted phosphoprotein 1 in silicotic rats.

Silicosis is caused by exposure to crystalline silica and the molecular mechanism of silicotic fibrosis remains unclear. Therefore, the present study investigated the mRNA profiles of rats exposed to crystalline silica. RNA-sequencing techniques were used to observe differential expression of mRNAs in silicotic rats induced by chronic inhalation of crystalline silica particulates. Prediction of mRNA functions and signaling pathways was conducted using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Certain differentially expressed mRNAs were verified in lung tissue of silicotic rats by quantitative polymerase chain reaction (qPCR). Secreted phosphoprotein 1 (SPP1) was measured in serum from silicosis patients, lungs of silicotic rats and NR8383 macrophages treated with silica. A total of 1,338 mRNAs were revealed to be differentially expressed in silicotic rat lungs, including 912 upregulated and 426 downregulated mRNAs. In GO analysis of significant changes in mRNAs, the most affected processes were the defense response, extracellular space and chemokine activity in terms of biological process, cellular component and molecular function. In KEGG pathway analysis, dysregulated mRNAs were involved in systemic lupus erythematosus, staphylococcus aureus infection, complement and coagulation cascades, alcoholism and pertussis. qPCR demonstrated that expression of Spp1, Mmp12, Ccl7, Defb5, Fabp4 and Slc26a4 was increased in silicotic rats, while Lpo, Itln1, Lcn2 and Dlk1 expression was decreased. It was also found that SPP1 was increased in serum from silicosis patients, silicotic rats and silica-treated NR8383 macrophages. The expression of mRNAs was altered significantly in silicotic rats, which suggested that certain genes are novel targets for the diagnosis and treatment of silicosis.

N-Acetyl-Seryl-Asparyl-Lysyl-Proline regulates lung renin angiotensin system to inhibit epithelial-mesenchymal transition in silicotic mice.

Silicosis is a major public health concern with various contributing factors. The renin-angiotensin system (RAS)is a critical regulator in the pathogenesis of this disease. We focused on two key RAS enzymes, angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2), to elucidate the activation of the ACE-angiotensin II (Ang II)-angiotensin II receptor 1 (AT1) axis and the inhibition of the ACE2-angiotensin-(1-7) [Ang-(1-7)]-Mas receptor axis in C57BL/6mice following SiO2 treatment. Silica exposure caused nodule formation, pulmonary interstitial fibrosis, epithelial-mesenchymal transition (EMT), abnormal deposition of extracellular matrix, and impaired lung function in mice. These effects were attenuated by the inhibition of ACE (captopril), blockade of the AT1(losartan), or systemic knockdown of the Ace gene. These effects were exacerbated by the inhibition of ACE2 (MLN-4760), blockade of the Mas (A779), or knockdown of the Ace2 gene. N-Acetyl-Seryl-Asparyl-Lysyl-Proline (Ac-SDKP), an anti-fibrotic peptide, ameliorated the silica-exposure-induced pathological changes by targeting the RAS system by activating the protective ACE2-Ang-(1-7)-Mas axis and inhibiting the deleterious ACE-Ang II-AT1 axis, thereby exerting a protective effect. This was confirmed in mouse lung type II epithelial cells (MLE-12) pretreated with Ang II and/or gene silencing separately targeting Ace and Ace2.The effects of Ac-SDKP were similar to those produced by Ace gene silencing and were partly attenuated by Ace2 deficiency. These findings suggested that RAS plays critical roles in the pathomechanism of silicosis fibrosis and that Ac-SDKP regulates lung RAS to inhibit EMT in silicotic mice and MLE-12 cells.

Pulmonary Silicosis Alters MicroRNA Expression in Rat Lung and miR-411-3p Exerts Anti-fibrotic Effects by Inhibiting MRTF-A/SRF Signaling.

To identify potential therapeutic targets for pulmonary fibrosis induced by silica, we studied the effects of this disease on the expression of microRNAs (miRNAs) in the lung. Rattus norvegicus pulmonary silicosis models were used in conjunction with high-throughput screening of lung specimens to compare the expression of miRNAs in control and pulmonary silicosis tissues. A total of 70 miRNAs were found to be differentially expressed between control and pulmonary silicosis tissues. This included 41 miRNAs that were upregulated and 29 that were downregulated relative to controls. Among them, miR-292-5p, miR-155-3p, miR-1193-3p, miR-411-3p, miR-370-3p, and miR-409a-5p were found to be similarly altered in rat lung and transforming growth factor (TGF)-ß1-induced cultured fibroblasts. Using miRNA mimics and inhibitors, we found that miR-1193-3p, miR-411-3p, and miR-370-3p exhibited potent anti-fibrotic effects, while miR-292-5p demonstrated pro-fibrotic effects in TGF-ß1-stimulated lung fibroblasts. Moreover, we also found that miR-411-3p effectively reduced pulmonary silicosis in the mouse lung by regulating Mrtfa expression, as demonstrated using biochemical and histological assays. In conclusion, our findings indicate that miRNA expression is perturbed in pulmonary silicosis and suggest that therapeutic interventions targeting specific miRNAs might be effective in the treatment of this occupational disease.

ACE2 Attenuates Epithelial-Mesenchymal Transition in MLE-12 Cells Induced by Silica.

PURPOSE: The role of angiotensin-converting enzyme 2 (ACE2) in silicosis remains unknown, although previous studies have suggested that ACE2 may be beneficial. We, therefore, investigated the effect of ACE2 on silicosis, particularly with regard to its role in regulating the epithelial-mesenchymal transition (EMT) induced by silica, with the aim to uncover a new potential target for the treatment of pulmonary fibrosis. MATERIALS AND METHODS: We employed wild-type mice treated with diminazene aceturate (DIZE, an ACE2 activator, 15 mg/kg/day for 4 weeks), hACE2-transgenic mice (overexpress the ACE2 gene), and the mouse lung type II epithelial cell line treated with DIZE (10-7 M for 48 h) or angiotensin-(1-7) [Ang-(1-7)] (10-4 M for 48 h), following induced fibrotic responses to determine the protective potential of ACE2. Silicosis models were established by orotracheal instillation of SiO2 (2.5 mg/mouse). Immunostaining was used to determine α-smooth muscle actin (α-SMA) expression. The activities of angiotensin-converting enzyme (ACE) and ACE2 and the levels of angiotensin II (Ang II) and Ang-(1-7) were detected by enzyme-linked immunosorbent assay. The mRNA expression of ACE and ACE2, and protein expression of the renin-angiotensin system (RAS) components and EMT indicators were studied by qRT-PCR and Western blot, respectively. RESULTS: DIZE treatment and overexpression of ACE2 markedly inhibited the formation of silica-induced lung fibrosis and increased the level of E-cadherin, with concomitant downregulation of pro-collagen, vimentin, and α-SMA via RAS signaling. Furthermore, DIZE and Ang-(1-7) attenuated the EMT and collagen deposition induced by silica in MLE-12 cells. Moreover, these effects were abrogated by MLN-4760 (a specific ACE2 inhibitor) and A779 (a specific Mas receptor blocker). CONCLUSION: The overexpression of ACE2 and treatment with DIZE can ameliorate EMT in silicotic mice via activation of the ACE2-Ang-(1-7)-Mas receptor axis, and these changes are accompanied by suppression of the ACE-Ang II-AT1 receptor axis.