Inhibition of STAT3 by S3I-201 suppress peritoneal fibroblast phenotype conversion and alleviate peritoneal fibrosis.

Peritoneal fibrosis is a common pathological response to long-term peritoneal dialysis (PD) and a major cause for PD discontinuation. Understanding the cellular and molecular mechanisms underlying the induction and progression of peritoneal fibrosis is of great interest. In our study, in vitro study revealed that signal transducer and activator of transcription 3 (STAT3) is a key factor in fibroblast activation and extracellular matrix (ECM) synthesis. Furthermore, STAT3 induced by IL-6 trans-signalling pathway mediate the fibroblasts of the peritoneal stroma contributed to peritoneal fibrosis. Inhibition of STAT3 exerts an antifibrotic effect by attenuating fibroblast activation and ECM production with an in vitro co-culture model. Moreover, STAT3 plays an important role in the peritoneal fibrosis in an animal model of peritoneal fibrosis developed in mice. Blocking STAT3 can reduce the peritoneal morphological changes induced by chlorhexidine gluconate. In conclusion, our findings suggested STAT3 signalling played an important role in peritoneal fibrosis. Therefore, blocking STAT3 might become a potential treatment strategy in peritoneal fibrosis.

Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

Fibroblasts' secretome from calcified and non-calcified dermis in Pseudoxanthoma elasticum differently contributes to elastin calcification.

Pseudoxanthoma elasticum (PXE) is a rare disease characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with this genetic metabolic condition, it is not known why elastic fibers in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome are investigated in vitro in CUS and CAS fibroblasts. Here we show that, compared to CUS, CAS fibroblasts exhibit: a) differently distributed and organized focal adhesions and stress fibers; b) modified cell-matrix interactions (i.e., collagen gel retraction); c) imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases; d) differentially expressed pro- and anti-calcifying proteoglycans and elastic-fibers associated glycoproteins. These data emphasize that in the development of pathologic mineral deposition fibroblasts play an active role altering the stability of elastic fibers and of the extracellular matrix milieu creating a local microenvironment guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component. In conclusion, this study contributes to a better understanding of the mechanisms of the mineral deposition that can be also associated with several inherited or age-related diseases (e.g., diabetes, atherosclerosis, chronic kidney diseases).

A novel MT-ATP6 variant associated with complicated ataxia in two unrelated Italian patients: case report and functional studies.

BACKGROUND: MT-ATP6 is a mitochondrial gene which encodes for the intramembrane subunit 6 (or A) of the mitochondrial ATP synthase, also known asl complex V, which is involved in the last step of oxidative phosphorylation to produce cellular ATP through aerobic metabolism. Although classically associated with the NARP syndrome, recent evidence highlights an important role of MT-ATP6 pathogenic variants in complicated adult-onset ataxias. METHODS: We describe two unrelated patients with adult-onset cerebellar ataxia associated with severe optic atrophy and mild cognitive impairment. Whole mitochondrial DNA sequencing was performed in both patients. We employed patients' primary fibroblasts and cytoplasmic hybrids (cybrids), generated from patients-derived cells, to assess the activity of respiratory chain complexes, oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential. RESULTS: In both patients, we identified the same novel m.8777 T > C variant in MT-ATP6 with variable heteroplasmy level in different tissues. We identifed an additional heteroplasmic novel variant in MT-ATP6, m.8879G > T, in the patients with the most severe phenotype. A significant reduction in complex V activity, OCR and ATP production was observed in cybrid clones homoplasmic for the m.8777 T > C variant, while no functional defect was detected in m.8879G > T homoplasmic clones. In addition, fibroblasts with high heteroplasmic levelsof m.8777 T > C variant showed hyperpolarization of mitochondrial membranes. CONCLUSIONS: We describe a novel pathogenic mtDNA variant in MT-ATP6 associated with adult-onset ataxia, reinforcing the value of mtDNA screening within the diagnostic workflow of selected patients with late onset ataxias.

Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Pirfenidone and nintedanib exert additive antifibrotic effects by the SPP1-AKT pathway in macrophages and fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with high mortality rates. It has been shown that pirfenidone (PFD) and nintedanib (Ofev) can slow down the decline in lung function of IPF patients, but their efficacy remains suboptimal. Some studies have suggested that the combination of PFD and Ofev may yield promising results. However, there is a lack of research on the combined application of these two medications in the treatment of IPF. A mouse model of bleomycin-induced (BLM) pulmonary fibrosis was established to investigate the impact of combination therapy on pulmonary fibrosis of mice. The findings demonstrated a significant reduction in lung tissue damage in mice treated with the combination therapy. Subsequent transcriptome analysis identified the differential gene secreted phosphoprotein 1 (SPP1), which was found to be associated with macrophages and fibroblasts based on multiple immunofluorescence staining results. Analysis of a phosphorylated protein microarray indicated that SPP1 plays a regulatory role in macrophages and fibroblasts via the AKT pathway. Consequently, the regulation of macrophages and fibroblasts in pulmonary fibrosis by the combination of PFD and Ofev is mediated by SPP1 through the AKT pathway, potentially offering a novel therapeutic option for IPF patients. Further investigation into the targeting of SPP1 for the treatment of pulmonary fibrosis is warranted.

Sox9-coordinated cellular neighborhoods generate fibrosis.

Poorly regenerative organs deposit scar tissue to mend damage. Aggarwal et al. establish that transient Sox9 activity is necessary for early proximal tubule epithelial regeneration, while Trogisch et al. and Aggarwal et al. show that persistent Sox9 activity in epithelial and endothelial cells activates fibroblasts creating fibrotic microdomains in multiple organs.

Targeting Interactions between Fibroblasts and Macrophages to Treat Cardiac Fibrosis.

Excessive extracellular matrix (ECM) deposition is a defining feature of cardiac fibrosis. Most notably, it is characterized by a significant change in the concentration and volume fraction of collagen I, a disproportionate deposition of collagen subtypes, and a disturbed ECM network arrangement, which directly affect the systolic and diastolic functions of the heart. Immune cells that reside within or infiltrate the myocardium, including macrophages, play important roles in fibroblast activation and consequent ECM remodeling. Through both direct and indirect connections to fibroblasts, monocyte-derived macrophages and resident cardiac macrophages play complex, bidirectional, regulatory roles in cardiac fibrosis. In this review, we discuss emerging interactions between fibroblasts and macrophages in physiology and pathologic conditions, providing insights for future research aimed at targeting macrophages to combat cardiac fibrosis.

Modeling acute myocardial infarction and cardiac fibrosis using human induced pluripotent stem cell-derived multi-cellular heart organoids.

Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.

Targeting pathogenic fibroblast-like synoviocyte subsets in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLSs) play a central role in RA pathogenesis and are the main cellular component in the inflamed synovium of patients with rheumatoid arthritis (RA). FLSs are emerging as promising new therapeutic targets in RA. However, fibroblasts perform many essential functions that are required for sustaining tissue homeostasis. Direct targeting of general fibroblast markers on FLSs is challenging because fibroblasts in other tissues might be altered and side effects such as reduced wound healing or fibrosis can occur. To date, no FLS-specific targeted therapies have been applied in the clinical management of RA. With the help of high-throughput technologies such as scRNA-seq in recent years, several specific pathogenic FLS subsets in RA have been identified. Understanding the characteristics of these pathogenic FLS clusters and the mechanisms that drive their differentiation can provide new insights into the development of novel FLS-targeting strategies for RA. Here, we discuss the pathogenic FLS subsets in RA that have been elucidated in recent years and potential strategies for targeting pathogenic FLSs.

IQGAP1 Regulates Actin Polymerization and Contributes to Bleomycin-Induced Lung Fibrosis.

We previously found IQ motif containing GTPase activating protein (IQGAP1) to be consistently elevated in lung fibroblasts (LF) isolated from patients with scleroderma (systemic sclerosis, SSc)-associated interstitial lung disease (ILD) and reported that IQGAP1 contributed to SSc by regulating expression and organization of α-smooth muscle actin (SMA) in LF. The aim of this study was to compare the development of ILD in the presence and absence of IQGAP1. Pulmonary fibrosis was induced in IQGAP1 knockout (KO) and wild-type (WT) mice by a single-intratracheal instillation of bleomycin. Two and three weeks later, mice were euthanized and investigated. We observed that the IQGAP1 KO mouse was characterized by a reduced rate of actin polymerization with reduced accumulation of actin in the lung compared to the WT mouse. After exposure to bleomycin, the IQGAP1 KO mouse demonstrated decreased contractile activity of LF, reduced expression of SMA, TGFß, and collagen, and lowered overall fibrosis scores compared to the WT mouse. The numbers of inflammatory cells and expression of pro-inflammatory cytokines in lung tissue were not significantly different between IQGAP1 KO and WT mice. We conclude that IQGAP1 plays an important role in the development of lung fibrosis induced by bleomycin, and the absence of IQGAP1 reduces the contractile activity of lung fibroblast and bleomycin-induced pulmonary fibrosis. Thus, IQGAP1 may be a potential target for novel anti-fibrotic therapies for lung fibrosis.

Mesothelioma-Associated Fibroblasts Modulate the Response of Mesothelioma Patient-Derived Organoids to Chemotherapy via Interleukin-6.

Malignant pleural mesothelioma (MPM) remains an incurable disease. This is partly due to the lack of experimental models that fully recapitulate the complexity and heterogeneity of MPM, a major challenge for therapeutic management of the disease. In addition, the contribution of the MPM microenvironment is relevant for the adaptive response to therapy. We established mesothelioma patient-derived organoid (mPDO) cultures from MPM pleural effusions and tested their response to pemetrexed and cisplatin. We aimed to evaluate the contribution of mesothelioma-associated fibroblasts (MAFs) to the response to pemetrexed and cisplatin (P+C). Organoid cultures were obtained from eight MPM patients using specific growth media and conditions to expand pleural effusion-derived cells. Flow cytometry was used to verify the similarity of the organoid cultures to the original samples. MAFs were isolated and co-cultured with mPDOs, and the addition of MAFs reduced the sensitivity of mPDOs to P+C. Organoid formation and expression of cancer stem cell markers such as ABCG2, NANOG, and CD44 were altered by conditioned media from treated MAFs. We identified IL-6 as the major contributor to the attenuated response to chemotherapy. IL-6 secretion by MAFs is correlated with increased resistance of mPDOs to pemetrexed and cisplatin.

Age-Dependent RGS5 Loss in Pericytes Induces Cardiac Dysfunction and Fibrosis.

BACKGROUND: Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. METHODS: In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. RESULTS: Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFß (transforming growth factor beta)2-dependent mechanism. CONCLUSIONS: Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.

Macrophage-fibroblast JAK/STAT dependent crosstalk promotes liver metastatic outgrowth in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease for which better therapies are urgently needed. Fibroblasts and macrophages are heterogeneous cell populations able to enhance metastasis, but the role of a macrophage-fibroblast crosstalk in regulating their pro-metastatic functions remains poorly understood. Here we deconvolve how macrophages regulate metastasis-associated fibroblast (MAF) heterogeneity in the liver. We identify three functionally distinct MAF populations, among which the generation of pro-metastatic and immunoregulatory myofibroblastic-MAFs (myMAFs) critically depends on macrophages. Mechanistically, myMAFs are induced through a STAT3-dependent mechanism driven by macrophage-derived progranulin and cancer cell-secreted leukaemia inhibitory factor (LIF). In a reciprocal manner, myMAF secreted osteopontin promotes an immunosuppressive macrophage phenotype resulting in the inhibition of cytotoxic T cell functions. Pharmacological blockade of STAT3 or myMAF-specific genetic depletion of STAT3 restores an anti-tumour immune response and reduces metastases. Our findings provide molecular insights into the complex macrophage-fibroblast interactions in tumours and reveal potential targets to inhibit PDAC liver metastasis.

Distinct fibroblast functions associated with fibrotic and immune-mediated inflammatory diseases and their implications for therapeutic development.

Fibroblasts are ubiquitous cells that can adopt many functional states. As tissue-resident sentinels, they respond to acute damage signals and shape the earliest events in fibrotic and immune-mediated inflammatory diseases. Upon sensing an insult, fibroblasts produce chemokines and growth factors to organize and support the response. Depending on the size and composition of the resulting infiltrate, these activated fibroblasts may also begin to contract or relax thus changing local stiffness within the tissue. These early events likely contribute to the divergent clinical manifestations of fibrotic and immune-mediated inflammatory diseases. Further, distinct changes to the cellular composition and signaling dialogue in these diseases drive progressive fibroblasts specialization. In fibrotic diseases, fibroblasts support the survival, activation and differentiation of myeloid cells, granulocytes and innate lymphocytes, and produce most of the pathogenic extracellular matrix proteins. Whereas, in immune-mediated inflammatory diseases, sequential accumulation of dendritic cells, T cells and B cells programs fibroblasts to support local, destructive adaptive immune responses. Fibroblast specialization has clear implications for the development of effective induction and maintenance therapies for patients with these clinically distinct diseases.

Single cell sequencing data identify distinct B cell and fibroblast populations in stricturing Crohn's disease.

Single cell RNA sequencing of human full thickness Crohn's disease (CD) small bowel resection specimens was used to identify potential therapeutic targets for stricturing (S) CD. Using an unbiased approach, 16 cell lineages were assigned within 14,539 sequenced cells from patient-matched SCD and non-stricturing (NSCD) preparations. SCD and NSCD contained identical cell types. Amongst immune cells, B cells and plasma cells were selectively increased in SCD samples. B cell subsets suggested formation of tertiary lymphoid tissue in SCD and compared with NSCD there was an increase in IgG, and a decrease in IgA plasma cells, consistent with their potential role in CD fibrosis. Two Lumican-positive fibroblast subtypes were identified and subclassified based on expression of selectively enriched genes as fibroblast clusters (C) 12 and C9. Cells within these clusters expressed the profibrotic genes Decorin (C12) and JUN (C9). C9 cells expressed ACTA2; ECM genes COL4A1, COL4A2, COL15A1, COL6A3, COL18A1 and ADAMDEC1; LAMB1 and GREM1. GO and KEGG Biological terms showed extracellular matrix and stricture organization associated with C12 and C9, and regulation of WNT pathway genes with C9. Trajectory and differential gene analysis of C12 and C9 identified four sub-clusters. Intra sub-cluster gene analysis detected 13 co-regulated gene modules that aligned along predicted pseudotime trajectories. CXCL14 and ADAMDEC1 were key markers in module 1. Our findings support further investigation of fibroblast heterogeneity and interactions with local and circulating immune cells at earlier time points in fibrosis progression. Breaking these interactions by targeting one or other population may improve therapeutic management for SCD.

The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition.

BACKGROUND: Renal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood. METHODS: To investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts. RESULTS: In this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis. CONCLUSIONS: Our findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD.

TRPM7 facilitates fibroblast-like synoviocyte proliferation, metastasis and inflammation through increasing IL-6 stability via the PKCα-HuR axis in rheumatoid arthritis.

Transient receptor potential melastatin 7 (TRPM7) is a cation channel that plays a role in the progression of rheumatoid arthritis (RA), yet its involvement in synovial hyperplasia and inflammation has not been determined. We previously reported that TRPM7 affects the destruction of articular cartilage in RA. Herein, we further confirmed the involvement of TRPM7 in fibroblast-like synoviocyte (FLS) proliferation, metastasis and inflammation. We observed increased TRPM7 expression in FLSs derived from human RA patients. Pharmacological inhibition of TRPM7 protected primary RA-FLSs from proliferation, metastasis and inflammation. Furthermore, we found that TRPM7 contributes to RA-FLS proliferation, metastasis and inflammation by increasing the intracellular Ca2+ concentration. Mechanistically, the PKCα-HuR axis was demonstrated to respond to Ca2+ influx, leading to TRPM7-mediated RA-FLS proliferation, metastasis and inflammation. Moreover, HuR was shown to bind to IL-6 mRNA after nuclear translocation, which could be weakened by TRPM7 channel inhibition. Additionally, adeno-associated virus 9-mediated TRPM7 silencing is highly effective at alleviating synovial hyperplasia and inflammation in adjuvant-induced arthritis rats. In conclusion, our findings unveil a novel regulatory mechanism involved in the pathogenesis of RA and suggest that targeting TRPM7 might be a potential strategy for the prevention and treatment of RA.

Curcumin regulates pulmonary extracellular matrix remodeling and mitochondrial function to attenuate pulmonary fibrosis by regulating the miR-29a-3p/DNMT3A axis.

Epigenetic regulation and mitochondrial dysfunction are essential to the progression of idiopathic pulmonary fibrosis (IPF). Curcumin (CCM) in inhibits the progression of pulmonary fibrosis by regulating the expression of specific miRNAs and pulmonary fibroblast mitochondrial function; however, the underlying mechanism is unclear. C57BL/6 mice were intratracheally injected with bleomycin (5 mg/kg) and treated with CCM (25 mg/kg body weight/3 times per week, intraperitoneal injection) for 28 days. Verhoeff-Van Gieson, Picro sirius red, and Masson's trichrome staining were used to examine the expression and distribution of collagen and elastic fibers in the lung tissue. Pulmonary fibrosis was determined using micro-computed tomography and transmission electron microscopy. Human pulmonary fibroblasts were transfected with miR-29a-3p, and RT-qPCR, immunostaining, and western blotting were performed to determine the expression of DNMT3A and extracellular matrix collagen-1 (COL1A1) and fibronectin-1 (FN1) levels. The expression of mitochondrial electron transport chain complex (MRC) and mitochondrial function were detected using western blotting and Seahorse XFp Technology. CCM in increased the expression of miR-29a-3p in the lung tissue and inhibited the DNMT3A to reduce the COL1A1 and FN1 levels leading to pulmonary extracellular matrix remodeling. In addition, CCM inhibited pulmonary fibroblasts MRC and mitochondrial function via the miR-29a-3p/DNMT3A pathway. CCM attenuates pulmonary fibrosis via the miR-29a-3p/DNMT3A axis to regulate extracellular matrix remodeling and mitochondrial function and may provide a new therapeutic intervention for preventing pulmonary fibrosis.