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Updated: Sep 20, 2025
Establishment and Validation of a Rat Model of Pulmonary Arterial Hypertension Associated with Pulmonary Fibrosis
Published on: May 23, 2025
Downregulated miR-129-5p expression inhibits rat pulmonary fibrosis by upregulating STAT1 gene expression in
Qingzeng Qian1, Qinghua Ma2, Bin Wang3
1School of Public Health, North China University of Science and Technology, Tangshan 063210, Hebei, China.
Objective:
This study investigated the mechanism by which microRNA-129-5p (miR-129-5p) in macrophages affects pulmonary fibrosis in rats by regulating the expression of the signal transducer and activator of transcription 1 (STAT1) gene.
Methods:
After the establishment of a pulmonary fibrosis rat model, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the expression of miR-129-5p in the sham group and model group. The binding sites between miR-129-5p and STAT1 were predicted online and verified by using a dual luciferase reporter system. qRT-PCR and Western blot analyses were used to test the effect of miR-129-5p on STAT1 gene expression. M2 macrophages were isolated and induced, and exosomes were extracted. Cell proliferation was detected by EdU. Furthermore, qRT-PCR was performed to detect the expression of STAT1, collagen type I A2 (COL1A2), collagen type III A1 (COL3A1), fibronectin, and α-SMA in cells and tissues followed by the detection of CD9, CD63, CD81, CD31 and STAT1 protein expression using a Western blot analysis. The pulmonary fibrosis area was detected by Masson staining followed by the immunohistochemical detection of α-smooth muscle actin (α-SMA) and type I collagen (COL-I) expression in pulmonary fibroblasts.
Results:
Compared with the sham group, the expression level of miR-129-5p in the model group was significantly increased (P < 0.05). miR-129-5p was observed to negatively regulate the expression of STAT1 (P < 0.05). The in vitro cell transfection experiments showed that after inhibiting the expression of miR-129-5p, the expression of STAT1 was increased, and the proliferation of fibroblasts and pulmonary fibrosis were inhibited (all P < 0.05). Furthermore, compared with the fibroblasts without coculture, the proliferation of the fibroblasts cocultured with M2 macrophage-secreted exosomes was clearly increased, and the expression levels of COL1A2, COL3A1, fibronectin and α-SMA were significantly increased (all P < 0.05). Compared with the mimic NC-exo group, the miR-129-5p-exo group had significantly increased proliferation of fibroblasts, decreased expression of STAT1, and significantly increased expression of COL1A2, COL3A1, fibronectin and α-SMA, and M2 macrophage-secreted exosomes could carry miR-129-5p to fibroblasts. Furthermore, the in vivo experiment confirmed that the exosomes of M2 macrophages could carry miR-129-5p, which could regulate M2 macrophages with pulmonary fibrosis in vivo.
Conclusion:
M2 macrophages can carry miR-129-5p to pulmonary interstitial fibroblasts and inhibit STAT1 gene expression, which may lead to the proliferation of fibroblasts and promote pulmonary fibrosis. The downregulation of miR-129-5p can significantly promote STAT1 gene expression in macrophages to inhibit pulmonary fibrosis in rats.
Insights
MicroRNA-129-5p (miR-129-5p) carried by M2 macrophage exosomes inhibits pulmonary fibrosis by downregulating STAT1. Downregulating miR-129-5p promotes STAT1 expression, thus inhibiting lung fibrosis in rats.
Area of Science:
- Cell Biology
- Molecular Biology
- Pulmonary Medicine
Background:
- Pulmonary fibrosis is a chronic lung disease characterized by excessive extracellular matrix deposition.
- The role of microRNAs (miRNAs) in regulating macrophage function and their impact on fibrotic diseases is an area of active investigation.
- Signal transducer and activator of transcription 1 (STAT1) is a transcription factor implicated in inflammatory and fibrotic processes.
Purpose of the Study:
- To elucidate the mechanism by which microRNA-129-5p (miR-129-5p) in macrophages influences pulmonary fibrosis in rats.
- To investigate the regulatory relationship between miR-129-5p and the signal transducer and activator of transcription 1 (STAT1) gene in the context of pulmonary fibrosis.
- To determine the role of M2 macrophage-derived exosomes in the intercellular transfer of miR-129-5p and its effect on pulmonary fibroblasts.
Main Methods:
- Establishment of a rat model of pulmonary fibrosis.
- Quantitative real-time polymerase chain reaction (qRT-PCR) to measure miR-129-5p and STAT1 expression.
- Dual luciferase reporter assay to confirm the binding sites between miR-129-5p and STAT1.
- Western blot analysis to assess protein expression levels.
- Isolation and induction of M2 macrophages, followed by exosome extraction and characterization.
- In vitro cell proliferation assays (EdU) and co-culture experiments with M2 macrophage-derived exosomes.
- Histological analysis (Masson staining) and immunohistochemistry for fibrotic markers (α-SMA, COL-I).
Main Results:
- miR-129-5p expression was significantly upregulated in the pulmonary fibrosis model group compared to the sham group.
- miR-129-5p was confirmed to negatively regulate STAT1 expression.
- Inhibition of miR-129-5p in vitro led to increased STAT1 expression, reduced fibroblast proliferation, and ameliorated pulmonary fibrosis.
- M2 macrophage-secreted exosomes were shown to carry miR-129-5p and transfer it to pulmonary fibroblasts, increasing fibroblast proliferation and extracellular matrix production (COL1A2, COL3A1, fibronectin, α-SMA).
- In vivo experiments confirmed that M2 macrophage exosomes carrying miR-129-5p could regulate pulmonary fibrosis.
Conclusions:
- M2 macrophages can transfer miR-129-5p via exosomes to pulmonary interstitial fibroblasts, inhibiting STAT1 expression and promoting fibroblast proliferation, thereby exacerbating pulmonary fibrosis.
- Downregulation of miR-129-5p in macrophages can enhance STAT1 gene expression, offering a potential therapeutic strategy to inhibit pulmonary fibrosis in rats.
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