Fine particulate matter (PM2.5) has been widely reported to contribute to the pathogenesis of pulmonary diseases. The direct hazardous effect of PM2.5 on the respiratory system at high concentrations in vitro and in vivo have been well identified. However, its effect on the pre-existing respiratory diseases of patients at environment-related concentrations remains unclear. Diesel exhaust PM2.5 as a primary representative of ambient PM2.5 fine particles were used to investigated the effect of PM2.5 on the fibrosis progression of existing pulmonary fibrosis disease models. This study reported that PM2.5 could result in the enhanced sensitivity to fibrotic response, which may be ascribed to ferroptosis induced by PM2.5 in damaged lung areas. Proteomic analysis revealed that the upregulation of HO-1 as a key mechanism in the ferroptosis and exacerbation of pulmonary fibrosis induced by PM2.5. As a result, HO-1 degraded heme-containing protein and released iron in fibrotic cells, leading to generation of mitochondrial ROS and impaired mitochondrial function. Transmission electron microscopic assay verified that PM2.5 entered the mitochondria of fibrotic cells and was accompanied by significant mitochondrial morphological changes characterized by increased mitochondrial membrane density and reduced mitochondrial size. The HO-1 inhibitor zinc protoporphyrin and mitochondrion-targeted antioxidant Mito-TEMPO significantly attenuated PM2.5-induced ferroptosis and exacerbation of fibrosis. In addition, AMPK-ULK1 axis-triggered autophagy activation and NCOA4-mediated degradation of ferritin by autophagy were found to be related to the PM2.5-induced ferroptosis of fibrotic cells. As evidenced by the inhibition of autophagy with 3-methyladenine or AMPK inhibitor, NCOA4 knockdown decreased intracellular iron accumulation and lipid peroxidation, thereby relieving PM2.5-induced epithelial-mesenchymal transition and cell death in fibrotic cells. Overall, this study provided experimental support for the idea that PM2.5 greatly deteriorates fibrosis process in pre-existing pulmonary fibrosis, and HO-1-mediated mitochondrial dysfunction and NCOA4-mediated ferritinophagy are jointly required for the PM2.5-induced ferroptosis and enhanced fibrosis effects.

Idiopathic pulmonary fibrosis (IPF) is characterized by lung scarring and has no effective treatment. Fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration are major clinical manifestations of this disease; hence, blocking these processes is a practical treatment strategy. Here, highly upregulated LINC00941/lncIAPF was found to accelerate pulmonary fibrosis by promoting fibroblast-to-myofibroblast differentiation and myofibroblast proliferation and migration. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation experiments elucidated that histone 3 lysine 27 acetylation (H3K27ac) activated the chromosome region opening in the LINC00941 promoter. As a consequence, the transcription factor ATF3 (activating transcription factor 3) bound to this region, and LINC00941 transcription was enhanced. RNA affinity isolation, RNA immunoprecipitation (RIP), RNase-RIP, half-life analysis, and ubiquitination experiments unveiled that LINC00941 formed a RNA-protein complex with ELAVL1/HuR (ELAV like RNA binding protein 1) to exert its pro-fibrotic function. Dual-fluorescence mRFP-GFP-MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) adenovirus monitoring technology, human autophagy RT² profiler PCR array, and autophagic flux revealed that the LINC00941-ELAVL1 axis inhibited autophagosome fusion with a lysosome. ELAVL1 RIP-seq, RIP-PCR, mRNA stability, and rescue experiments showed that the LINC00941-ELAVL1 complex inhibited autophagy by controlling the stability of the target genes EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), STAT1 (signal transducer and activators of transcription 1) and FOXK1 (forkhead box K1). Finally, the therapeutic effect of LINC00941 was confirmed in a mouse model and patients with IPF. This work provides a therapeutic target and a new effective therapeutic strategy related to autophagy for IPF.

Noncoding RNAs (ncRNAs), such as microRNA (miRNA), long ncRNA (lncRNA), and circular RNA (circRNA), are regulators of important biological functions. Therefore, understanding their crosstalk and regulatory patterns can provide treatment for diseases. In this study, differentially expressed RNA transcripts were obtained by RNA sequencing in bleomycin-induced pulmonary fibrosis in mice. Four miRNAs, 10 lncRNAs, and two circRNAs were tested to validate the sequencing. There were differentially expressed 585 mRNAs, 236 miRNAs, 272 lncRNAs, and 74 circRNAs in pulmonary fibrosis. Their location on chromosome, length varieties, interaction, and host genes were analyzed. lnc949, circ949, and circ057 were chosen to explore the detailed crosstalk and regulatory pattern, which were measured by using RNA-FISH, dual-luciferase reporter assay, real-time cell analysis and rescue experiment, co-localization analysis, RNA immunoprecipitation, and RNA pull down. The data showed that the three ncRNAs were predominant in the cytoplasm, and their regulatory patterns were focused on post-transcription. The fibrotic function of lnc949 depended on its host gene FKBP5. circ949 and circ057 formed a regulatory network with lnc865 and lnc556 to simultaneously regulate miR-29b-2-5p targeting STAT3 phosphorylation. Collectively, different RNAs can crosstalk with each other to regulate pulmonary fibrosis through different regulatory patterns. We hope these data can provide a full concept of RNA transcripts, leading to a new treatment for pulmonary fibrosis.

Promotion of myofibroblast apoptosis is a potential therapeutic strategy for pulmonary fibrosis. This study investigated the antifibrotic effect of astaxanthin on the promotion of myofibroblast apoptosis based on dynamin‐related protein‐1 (Drp1)‐mediated mitochondrial fission in vivo and in vitro. Results showed that astaxanthin can inhibit lung parenchymal distortion and collagen deposition, as well as promote myofibroblast apoptosis. Astaxanthin demonstrated pro‐apoptotic function in myofibroblasts by contributing to mitochondrial fission, thereby leading to apoptosis by increasing the Drp1 expression and enhancing Drp1 translocation into the mitochondria. Two specific siRNAs were used to demonstrate that Drp1 is necessary to promote astaxanthin‐induced mitochondrial fission and apoptosis in myofibroblasts. Drp1‐associated genes, such as Bcl‐2‐associated X protein, cytochrome c, tumour suppressor gene p53 and p53‐up‐regulated modulator of apoptosis, were highly up‐regulated in the astaxanthin group compared with those in the sham group. This study revealed that astaxanthin can prevent pulmonary fibrosis by promoting myofibroblast apoptosis through a Drp1‐dependent molecular pathway. Furthermore, astaxanthin provides a potential therapeutic value in pulmonary fibrosis treatment.

Emerging evidence suggests that microRNA (miRNA) and long noncoding RNA (lncRNA) play important roles in disease development. However, the mechanism underlying mRNA interaction with miRNA and lncRNA in idiopathic pulmonary fibrosis (IPF) remains unknown. This study presents a novel lnc-PCF that promotes the proliferation of TGF-β1-activated epithelial cells through the regulation of map3k11 by directly targeting miR-344a-5p during pulmonary fibrogenesis. Bioinformatics and in vitro translation assay were performed to confirm whether or not lnc-PCF is an actual lncRNA. RNA fluorescent in situ hybridization (FISH) and nucleocytoplasmic separation showed that lnc-PCF is mainly expressed in the cytoplasm. Knockdown and knockin of lnc-PCF indicated that lnc-PCF could promote fibrogenesis by regulating the proliferation of epithelial cells activated by TGF-β1 according to the results of xCELLigence real-time cell analysis system, flow cytometry, and western blot analysis. Computational analysis and a dual-luciferase reporter system were used to identify the target gene of miR-344a-5p, whereas RNA pull down, anti-AGO2 RNA immunoprecipitation, and rescue experiments were conducted to confirm the identity of this direct target. Further experiments verified that lnc-PCF promotes the proliferation of activated epithelial cells that were dependent on miR-344a-5p, which exerted its regulatory functions through its target gene map3k11. Finally, adenovirus packaging sh-lnc-PCF was sprayed into rat lung tissues to evaluate the therapeutic effect of lnc-PCF. These findings revealed that lnc-PCF can accelerate pulmonary fibrogenesis by directly targeting miR-344a-5p to regulate map3k11, which may be a potential therapeutic target in IPF.

MicroRNAs (miRNAs) are important diagnostic markers and therapeutic targets for many diseases. However, the miRNAs that control the pathogenesis of idiopathic pulmonary fibrosis (IPF) and act as potential therapeutic targets for the disease are rarely studied. In the present study, we analyzed the function and regulatory mechanism of microRNA-708-3p (miR-708-3p) and evaluated this marker’s potential as a therapeutic target in IPF. The clinical and biological relevance of fibrogenesis for miR-708-3p was assessed in vivo and in vitro, specifically in matching plasma and tissue samples from 78 patients with IPF. The data showed that the miR-708-3p levels decreased during fibrosis and inversely correlated with IPF. The experiments showed that the decreased miR-708 promoter activity and primer-miR-708(pri-miR-708) expression were the potential causes. By computational analysis, a dual luciferase reporter system, rescue experiments and a Cignal Finder 45-Pathway system with siADAM17 and a miR-708-3p mimic, we identified that miR-708-3p directly regulates its target gene, a disintegrin and metalloproteinase 17 (ADAM17), through a binding site in the 3′ untranslated region, which depends on the GATA/STAT3 signaling pathway. Finally, an miR-708-3p agomir was designed and used to test the therapeutic effects of the miR-708-3p in an animal model. Small-animal imaging technology and other experiments showed that the dynamic image distribution of the miR-708-3p agomir was mainly concentrated in the lungs and could block fibrogenesis. In conclusion, the miR-708-3p–ADAM17 axis aggravates IPF, and miR-708-3p can serve as a potential therapeutic target for IPF.