Aging-Associated Metabolite Methylmalonic Acid Increases Susceptibility to Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by pulmonary fibroblast overactivation, resulting in the accumulation of abnormal extracellular matrix and lung parenchymal damage. Although the pathogenesis of IPF remains unclear, aging was proposed as the most prominent nongenetic risk factor. Propionate metabolism undergoes reprogramming in the aging population, leading to the accumulation of the by-product methylmalonic acid (MMA). This study aimed to explore alterations in propionate metabolism in IPF and the impact of the by-product MMA on pulmonary fibrosis. It revealed alterations in the expression of enzymes involved in propionate metabolism within IPF lung tissues, characterized by an increase in propionyl-CoA carboxylase and methylmalonyl-CoA epimerase expression, and a decrease in methylmalonyl-CoA mutase expression. Knockdown of methylmalonyl-CoA mutase, the key enzyme in propionate metabolism, induced a profibrotic phenotype and activated co-cultured fibroblasts in A549 cells. MMA exacerbated bleomycin-induced mouse lung fibrosis and induced a profibrotic phenotype in both epithelial cells and fibroblasts through activation of the canonical transforming growth factor-ß/Smad pathway. Overall, these findings unveil an alteration of propionate metabolism in IPF, leading to MMA accumulation, thus exacerbating lung fibrosis through promoting profibrotic phenotypic transitions via the canonical transforming growth factor-ß/Smad signaling pathway.

Lactate accumulation induced by Akt2-PDK1 signaling promotes pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with an abnormal accumulation of fibrotic tissue in the lung parenchyma and elevated glycolysis level in associated cells without effective therapy options. Lactate accumulation in pulmonary fibrotic tissue is a significant factor aggravating IPF development, but the main mechanism regulating glycolysis needs further investigation. In this study, lung fibrosis model was induced by bleomycin (BLM) intratracheally in female C57BL/6 mice. The changes of lactate level and fibrotic markers were detected. For in vitro studies, cell lines of alveolar epithelial cell and lung fibroblast cell were stimulated with TGF-ß1 and BLM respectively, to detect changes in their fibrotic properties. The function of lactate accumulation on facilitating fibrosis was verified. We demonstrated that BLM-induced pulmonary fibrosis is accompanied by lactate accumulation owing to glycolysis upregulation. Significantly high PDK1 expression in lung fibrotic tissue promotes glycolysis. Moreover, PDK1 stimulated trans-differentiation of lung fibroblasts and epithelial-mesenchymal transition (EMT) of alveolar epithelial cells. Furthermore, phosphorylated Akt2 activated PDK1 to cause pulmonary fibrosis and inhibitors of Akt2 and PDK1 could suppress fibrotic process. This study is the first to consider PDK1 facilitated lactate accumulation through glycolysis as a vital factor in pulmonary fibrosis and could be initiated by Akt2. We concluded that the pro-fibrotic properties of PDK1 are associated with Akt2 phosphorylation and thus provide new potential therapeutic targets for pulmonary fibrosis.

Repair and regeneration of the alveolar epithelium in lung injury.

Considerable progress has been made in understanding the function of alveolar epithelial cells in a quiescent state and regeneration mechanism after lung injury. Lung injury occurs commonly from severe viral and bacterial infections, inhalation lung injury, and indirect injury sepsis. A series of pathological mechanisms caused by excessive injury, such as apoptosis, autophagy, senescence, and ferroptosis, have been studied. Recovery from lung injury requires the integrity of the alveolar epithelial cell barrier and the realization of gas exchange function. Regeneration mechanisms include the participation of epithelial progenitor cells and various niche cells involving several signaling pathways and proteins. While alveoli are damaged, alveolar type II (AT2) cells proliferate and differentiate into alveolar type I (AT1) cells to repair the damaged alveolar epithelial layer. Alveolar epithelial cells are surrounded by various cells, such as fibroblasts, endothelial cells, and various immune cells, which affect the proliferation and differentiation of AT2 cells through paracrine during alveolar regeneration. Besides, airway epithelial cells also contribute to the repair and regeneration process of alveolar epithelium. In this review, we mainly discuss the participation of epithelial progenitor cells and various niche cells involving several signaling pathways and transcription factors.

Lactate facilitated mitochondrial fission-derived ROS to promote pulmonary fibrosis via ERK/DRP-1 signaling.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung diseases, which mainly existed in middle-aged and elderly people. The accumulation of reactive oxygen species (ROS) is a common characteristic of IPF. Previous research also shown that lactate levels can be abnormally elevated in IPF patients. Emerging evidence suggested a relationship between lactate and ROS in IPF which needs further elucidation. In this article, we utilized a mouse model of BLM-induced pulmonary fibrosis to detect alterations in ROS levels and other indicators associated with fibrosis. Lactate could induce mitochondrial fragmentation by modulating expression and activity of DRP1 and ERK. Moreover, Increased ROS promoted P65 translocation into nucleus, leading to expression of lung fibrotic markers. Finally, Ulixertinib, Mdivi-1 and Mito-TEMPO, which were inhibitor activity of ERK, DRP1 and mtROS, respectively, could effectively prevented mitochondrial damage and production of ROS and eventually alleviate pulmonary fibrosis. Taken together, these findings suggested that lactate could promote lung fibrosis by increasing mitochondrial fission-derived ROS via ERK/DRP1 signaling, which may provide novel therapeutic solutions for IPF.

Downregulation of HMGCS2 mediated AECIIs lipid metabolic alteration promotes pulmonary fibrosis by activating fibroblasts.

BACKGROUND: Abnormal lipid metabolism has recently been reported as a crucial signature of idiopathic pulmonary fibrosis (IPF). However, the origin and biological function of the lipid and possible mechanisms of increased lipid content in the pathogenesis of IPF remains undetermined. METHODS: Oil-red staining and immunofluorescence analysis were used to detect lipid accumulation in mouse lung fibrosis frozen sections, Bleomycin-treated human type II alveolar epithelial cells (AECIIs) and lung fibroblast. Untargeted Lipid omics analysis was applied to investigate differential lipid species and identified LysoPC was utilized to treat human lung fibroblasts and mice. Microarray and single-cell RNA expression data sets identified lipid metabolism-related differentially expressed genes. Gain of function experiment was used to study the function of 3-hydroxy-3-methylglutaryl-Coa Synthase 2 (HMGCS2) in regulating AECIIs lipid metabolism. Mice with AECII-HMGCS2 high were established by intratracheally delivering HBAAV2/6-SFTPC- HMGCS2 adeno-associated virus. Western blot, Co-immunoprecipitation, immunofluorescence, site-directed mutation and flow cytometry were utilized to investigate the mechanisms of HMGCS2-mediated lipid metabolism in AECIIs. RESULTS: Injured AECIIs were the primary source of accumulated lipids in response to Bleomycin stimulation. LysoPCs released by injured AECIIs could activate lung fibroblasts, thus promoting the progression of pulmonary fibrosis. Mechanistically, HMGCS2 was decreased explicitly in AECIIs and ectopic expression of HMGCS2 in AECIIs using the AAV system significantly alleviated experimental mouse lung fibrosis progression via modulating lipid degradation in AECIIs through promoting CPT1A and CPT2 expression by interacting with PPARα. CONCLUSIONS: These data unveiled a novel etiological mechanism of HMGCS2-mediated AECII lipid metabolism in the genesis and development of pulmonary fibrosis and provided a novel target for clinical intervention.

NR2F2 alleviates pulmonary fibrosis by inhibition of epithelial cell senescence.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal, and aging-associated interstitial lung disease with a poor prognosis and limited treatment options, while the pathogenesis remains elusive. In this study, we found that the expression of nuclear receptor subfamily 2 group F member 2 (NR2F2), a member of the steroid thyroid hormone superfamily of nuclear receptors, was reduced in both IPF and bleomycin-induced fibrotic lungs, markedly in bleomycin-induced senescent epithelial cells. Inhibition of NR2F2 expression increased the expression of senescence markers such as p21 and p16 in lung epithelial cells, and activated fibroblasts through epithelial-mesenchymal crosstalk, inversely overexpression of NR2F2 alleviated bleomycin-induced epithelial cell senescence and inhibited fibroblast activation. Subsequent mechanistic studies revealed that overexpression of NR2F2 alleviated DNA damage in lung epithelial cells and inhibited cell senescence. Adenovirus-mediated Nr2f2 overexpression attenuated bleomycin-induced lung fibrosis and cell senescence in mice. In summary, these data demonstrate that NR2F2 is involved in lung epithelial cell senescence, and targeting NR2F2 may be a promising therapeutic approach against lung cell senescence and fibrosis.

Differential DNA methylation landscape of miRNAs genes in mice liver fibrosis.

BACKGROUND: Patients with chronic liver disease were found nearly all to have liver fibrosis, which is characterized by excess accumulation of extracellular matrix (ECM) proteins. While ECM accumulation can prevent liver infection and injury, it can destroy normal liver function and architecture. miRNA's own regulation was involved in DNA methylation change. The purpose of this study is to detect DNA methylation landscape of miRNAs genes in mice liver fibrosis tissues. METHODS: Male mice (10-12 weeks) were injected CCl4 from abdominal cavity to induced liver fibrosis. 850 K BeadChips were used to examine DNA methylation change in whole genome. The methylation change of 16 CpG dinucleotides located in promoter regions of 4 miRNA genes were detected by bisulfite sequencing polymerase chain reaction (BSP) to verify chip data accuracy, and these 4 miRNA genes' expressions were detected by RT-qPCR methods. RESULTS: There are 769 differential methylation sites (DMS) in total between fibrotic liver tissue and normal mice liver tissue, which were related with 148 different miRNA genes. Chips array data were confirmed by bisulfite sequencing polymerase chain reaction (R = 0.953; P < 0.01). GO analysis of the target genes of 2 miRNA revealed that protein binding, cytoplasm and chromatin binding activity were commonly enriched; KEGG pathway enrichment analysis displayed that TGF-beta signaling pathway was commonly enriched. CONCLUSION: The DNA of 148 miRNA genes was found to have methylation change in liver fibrosis tissue. These discoveries in miRNA genes are beneficial to future miRNA function research in liver fibrosis.

TRIOBP modulates ß-catenin signaling by regulation of miR-29b in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal and devastating lung disease of unknown etiology, described as the result of multiple cycles of epithelial cell injury and fibroblast activation. Despite this impressive increase in understanding, a therapy that reverses this form of fibrosis remains elusive. In our previous study, we found that miR-29b has a therapeutic effect on pulmonary fibrosis. However, its anti-fibrotic mechanism is not yet clear. Recently, our study identified that F-Actin Binding Protein (TRIOBP) is one of the target genes of miR-29b and found that deficiency of TRIOBP increases resistance to lung fibrosis in vivo. TRIOBP knockdown inhibited the proliferation of epithelial cells and attenuated the activation of fibroblasts. In addition, deficiency of Trio Rho Guanine Nucleotide Exchange Factor (TRIO) in epithelial cells and fibroblasts decreases susceptibility to lung fibrosis. TRIOBP interacting with TRIO promoted abnormal epithelial-mesenchymal crosstalk and modulated the nucleocytoplasmic translocation of ß-catenin. We concluded that the miR-29b‒TRIOBP-TRIO-ß-catenin axis might be a key anti-fibrotic axis in IPF to regulate lung regeneration and fibrosis, which may provide a promising treatment strategy for lung fibrosis.

Effect of LRRC15 on autophagy in A549 cells.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, and irreversible interstitial lung disease with unknown cause. To explore the role and regulatory mechanism of leucine-rich repeat-containing protein 15 (LRRC15) in IPF, bleomycin (BLM)-induced pulmonary fibrosis in mouse and A549 cells were constructed, and the expression of LRRC15 were detected. Then, MTT, GFP-RFP-LC3 dual fluorescent labeling system and Western blotting were used to investigate the effects of LRRC15 on cell activity and autophagy after transfection of siLRRC15, respectively. The results indicated that the expression of LRRC15 was significantly increased after the BLM treatment in mouse lung tissue and A549 cells. The designed and synthesized siLRRC15 followed by transfection into A549 cells resulted in a dramatic reduction in LRRC15 expression and partially restored the cell damage induced by BLM. Moreover, the expression of LC3-II and P62 were up-regulated, the amount of autophagosome were increased by GFP-RFP-LC3 dual fluorescent labeling assay after BLM treatment. Meanwhile, this study also showed that the key autophagy proteins LC3-II, ATG5 and ATG7 were up-regulated, P62 was down-regulated and autophagic flux were enhanced after further treatment of A549 cells with siLRRC15. The above findings suggest that LRRC15 is an indicator of epithelial cell damage and may participate in the regulation of fibrosis through autophagy mechanism in IPF. This study provides necessary theoretical basis for further elucidating the mechanism of IPF.

Tuftelin1 drives experimental pulmonary fibrosis progression by facilitating stress fiber assembly.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD) with unknown etiology, characterized by sustained damage repair of epithelial cells and abnormal activation of fibroblasts, the underlying mechanism of the disease remains elusive. METHODS: To evaluate the role of Tuftelin1 (TUFT1) in IPF and elucidate its molecular mechanism. We investigated the level of TUFT1 in the IPF and bleomycin-induced mouse models and explored the influence of TUFT1 deficiency on pulmonary fibrosis. Additionally, we explored the effect of TUFT1 on the cytoskeleton and illustrated the relationship between stress fiber and pulmonary fibrosis. RESULTS: Our results demonstrated a significant upregulation of TUFT1 in IPF and the bleomycin (BLM)-induced fibrosis model. Disruption of TUFT1 exerted inhibitory effects on pulmonary fibrosis in both in vivo and in vitro. TUFT1 facilitated the assembly of microfilaments in A549 and MRC-5 cells, with a pronounced association between TUFT1 and Neuronal Wiskott-Aldrich syndrome protein (N-WASP) observed during microfilament formation. TUFT1 can promote the phosphorylation of tyrosine residue 256 (Y256) of the N-WASP (pY256N-WASP). Furthermore, TUFT1 promoted transforming growth factor-ß1 (TGF-ß1) induced fibroblast activation by increasing nuclear translocation of pY256N-WASP in fibroblasts, while wiskostatin (Wis), an N-WASP inhibitor, suppressed these processes. CONCLUSIONS: Our findings suggested that TUFT1 plays a critical role in pulmonary fibrosis via its influence on stress fiber, and blockade of TUFT1 effectively reduces pro-fibrotic phenotypes. Pharmacological targeting of the TUFT1-N-WASP axis may represent a promising therapeutic approach for pulmonary fibrosis.

Glycolysis Reprogramming in Idiopathic Pulmonary Fibrosis: Unveiling the Mystery of Lactate in the Lung.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by excessive deposition of fibrotic connective tissue in the lungs. Emerging evidence suggests that metabolic alterations, particularly glycolysis reprogramming, play a crucial role in the pathogenesis of IPF. Lactate, once considered a metabolic waste product, is now recognized as a signaling molecule involved in various cellular processes. In the context of IPF, lactate has been shown to promote fibroblast activation, myofibroblast differentiation, and extracellular matrix remodeling. Furthermore, lactate can modulate immune responses and contribute to the pro-inflammatory microenvironment observed in IPF. In addition, lactate has been implicated in the crosstalk between different cell types involved in IPF; it can influence cell-cell communication, cytokine production, and the activation of profibrotic signaling pathways. This review aims to summarize the current research progress on the role of glycolytic reprogramming and lactate in IPF and its potential implications to clarify the role of lactate in IPF and to provide a reference and direction for future research. In conclusion, elucidating the intricate interplay between lactate metabolism and fibrotic processes may lead to the development of innovative therapeutic strategies for IPF.

Signaling pathways of liver regeneration: Biological mechanisms and implications.

The liver possesses a unique regenerative ability to restore its original mass, in this regard, partial hepatectomy (PHx) and partial liver transplantation (PLTx) can be executed smoothly and safely, which has important implications for the treatment of liver disease. Liver regeneration (LR) can be the very complicated procedure that involves multiple cytokines and transcription factors that interact with each other to activate different signaling pathways. Activation of these pathways can drive the LR process, which can be divided into three stages, namely, the initiation, progression, and termination stages. Therefore, it is important to investigate the pathways involved in LR to elucidate the mechanism of LR. This study reviews the latest research on the key signaling pathways in the different stages of LR.

Hibiscus manihot L. flower extract induces anticancer activity through modulation of apoptosis and autophagy in A549 cells.

Lung cancer is a major public health issue and heavy burden in China and worldwide due to its high incidence and mortality without effective treatment. It's imperative to develop new treatments to overcome drug resistance. Natural products from food source, given their wide-ranging and long-term benefits, have been increasingly used in tumor prevention and treatment. This study revealed that Hibiscus manihot L. flower extract (HML) suppressed the proliferation and migration of A549 cells in a dose and time dependent manner and disrupting cell cycle progression. HML markedly enhanced the accumulation of ROS, stimulated the dissipation of mitochondrial membrane potential (MMP) and that facilitated mitophagy through the loss of mitochondrial function. In addition, HML induced apoptosis by activation of the PTEN-P53 pathway and inhibition of ATG5/7-dependent autophagy induced by PINK1-mediated mitophagy in A549 cells. Moreover, HML exert anticancer effects together with 5-FU through synergistic effect. Taken together, HML may serve as a potential tumor prevention and adjuvant treatment for its functional attributes.

CCT6A alleviates pulmonary fibrosis by inhibiting HIF-1α-mediated lactate production.

Idiopathic pulmonary fibrosis (IPF) is a lethal progressive fibrotic lung disease. The development of IPF involves different molecular and cellular processes, and recent studies indicate that lactate plays a significant role in promoting the progression of the disease. Nevertheless, the mechanism by which lactate metabolism is regulated and the downstream effects remain unclear. The molecular chaperone CCT6A performs multiple functions in a variety of biological processes. Our research has identified a potential association between CCT6A and serum lactate levels in IPF patients. Herein, we found that CCT6A was highly expressed in type 2 alveolar epithelial cells (AEC2s) of fibrotic lung tissues and correlated with disease severity. Lactate increases the accumulation of lipid droplets in epithelial cells. CCT6A inhibits lipid synthesis by blocking the production of lactate in AEC2s and alleviates bleomycin-induced pulmonary fibrosis in mice. In addition, our results revealed that CCT6A blocks HIF-1α-mediated lactate production by driving the VHL-dependent ubiquitination and degradation of HIF-1α and further inhibits lipid accumulation in fibrotic lungs. In conclusion, we propose that there is a pivotal regulatory role of CCT6A in lactate metabolism in pulmonary fibrosis, and strategies aimed at targeting these key molecules could represent potential therapeutic approaches for pulmonary fibrosis.

Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs.

Background: Schistosomiasis is a zoonotic parasitic disorder induced by the infestation of schistosomes, a genus of trematodes. MicroRNAs (miRNAs) in egg-derived exosomes are crucial for modulating the host's immune responses and orchestrating the pathophysiological mechanisms. Although the exosomes secreted by S. japonicum contain abundant miRNAs, the specific roles of these miRNAs in the pathogenesis of schistosomiasis-induced hepatic fibrosis are yet to be comprehensively elucidated. The egg exosomes of S. japonicum secrete miRNA-30, a novel miRNA. Methods: In vitro, the effect of miRNA-30 was evaluated by transfecting HSCs with miRNA mimics. The target gene biosignature for miRNA-30 was predicted using the miRDB software. The effect of miRNA-30 in hepatic fibrosis was evaluated by either elevating its expression in healthy mice or by inhibiting its activity in infected mice by administration of recombinant adeno-associated virus serotype eight vectors expressing miRNA-30 or miRNA sponges. Results: This novel miRNA can activate hepatic stellate cells (HSCs), the prinary effector cells of hepatic fibrosis, in vitro, i.e., it significantly increases the fibrogenic factors Col1(α1), Col3(α1), and α-SMA at both mRNA and protein levels. In addition, miRNA-30 may activate HSCs by targeting the host RORA gene. In addition, in vivo experiments were conducted by administering a recombinant adeno-associated viral vector to modulate the expression levels of miRNA-30. The overexpression of miRNA-30 in healthy mice significantly elevated the expression of Col1(α1), Col3(α1), and α-SMA at both the transcriptomic and proteomic scales. This overexpression was coupled with a pronounced augmentation in the hepatic hydroxyproline content. Conversely, the in vivo silencing of miRNA-30 in infected mice induced a considerable reduction in the size of hepatic granulomas and areas of collagen deposition. Hence, in vivo, modulation of miRNA-30 expression may play a pivotal role in ameliorating the severity of hepatic fibrosis in mice afflicted with S. japonica. Conclusions: The study results suggest that miRNA-30 may augment schistosomiasis-induced hepatic fibrosis through a probable interaction with the host RORA. Our study may improve the current theoretical framework regarding cross-species regulation by miRNAs of hepatic fibrosis in schistosomiasis.

Comprehensive proteomic characterization of urethral stricture disease in the Chinese population.

Background: Male urethral stricture disease (USD) is predominantly characterized by scar formation. There are few effective therapeutic drugs, and comprehensive molecular characterizations of USD formation remain undefined. Methods: The proteomic profiling of twelve scar tissues and five matched normal adjacent tissues (NATs). Proteomic analysis methods were applied to explore the molecular characterizations of USD formation, including uncovering mechanistic pathways and providing novel biomarkers for scar formation. Results: Comparative proteomic analysis showed that the extracellular matrix (ECM) and complement cascade signaling were predominant in scar tissues. COL11A1 and CD248 significantly contributed to the accumulation of ECM components. Our study presented diverse molecular mechanisms of scar formation across different ages and suggested the potential effects of PXK in Age 1 (<45) patients. Furthermore, immune infiltration studies indicated the therapeutic potential of inhibiting the complement system (C4A, C4B) in Age 2 (≥45) patients, providing a potential clinical strategy for USD. Conclusion: This study illustrated the pathogenesis of USD formation and the diverse characteristics of USD patients with different ages, enhancing our understanding of the disease's pathogenesis and providing a valuable resource for USD treatment.

Proteomic Profiling of Serum Extracellular Vesicles Identifies Diagnostic Signatures and Therapeutic Targets in Breast Cancer.

Analysis of extracellular vesicles (EVs) is a promising noninvasive liquid biopsy approach for breast cancer (BC) detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of BC-specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry-based, data-independent acquisition (DIA) proteomic approach to characterize human serum EVs derived from patients with BC (n = 126) and healthy donors (HDs, n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled construction of specific EV protein classifiers for diagnosing BC and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of BC. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating BC invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit BC migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of BC patients to guide development of improved diagnosis, monitoring, and treatment strategies.