Pulmonary Fibrosis In Mice Research Articles

Caffeic acid ethanolamide induces antifibrosis, anti-inflammatory, and antioxidant effects protects against bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease; its cause is unknown, and it leads to notable health problems. Currently, only two drugs are recommended for IPF treatment. Although these drugs can mitigate lung function decline, neither can improve nor stabilize IPF or the symptoms perceived by patients. Therefore, the development of novel treatment options for pulmonary fibrosis is required. The present study investigated the effects of a novel compound, caffeic acid ethanolamide (CAEA), on human pulmonary fibroblasts and evaluated its potential to mitigate bleomycin-induced pulmonary fibrosis in mice. CAEA inhibited TGF-β-induced α-SMA and collagen expression in human pulmonary fibroblasts, indicating that CAEA prevents fibroblasts from differentiating into myofibroblasts following TGF-β exposure. In animal studies, CAEA treatment efficiently suppressed immune cell infiltration and the elevation of TNF-α and IL-6 in bronchoalveolar lavage fluid in mice with bleomycin-induced pulmonary fibrosis. Additionally, CAEA exerted antioxidant effects by recovering the enzymatic activities of oxidant scavengers. CAEA directly inhibited activation of TGF-β receptors and protected against bleomycin-induced pulmonary fibrosis through inhibition of the TGF-β/SMAD/CTGF signaling pathway. The protective effect of CAEA was comparable to that of pirfenidone, a clinically available drug. Our findings support the potential of CAEA as a viable method for preventing the progression of pulmonary fibrosis.

Cuproptosis-associated hub gene identification and immune cell infiltration patterns in silicosis

Recent research has hinted at a potential connection between silicosis, a fibrotic lung disease caused by exposure to crystalline silica particles, and cuproptosis. The aim of the study was to explore how cuproptosis-related genes (CRGs) may influence the development of silicosis and elucidate the underlying mechanisms. An analysis of genes associated with both silicosis and cuproptosis was conducted. Key gene identification was achieved through the application of two machine learning techniques. Additionally, the correlation between these key genes and immune cell populations was explored and the critical pathways were discerned. To corroborate our findings, the expression of key genes was verified in both a publicly available silica-induced mouse model and our own silicosis mouse model. A total of 12 differentially expressed CRGs associated with silicosis were identified. Further analysis resulted in the identification of 6 CRGs, namely LOX, SPARC, MOXD1, ALB, MT-CO2, and AOC2. Elevated immune cell infiltration of CD8 T cells, regulatory T cells, M0 macrophages, and neutrophils in silicosis patients compared to healthy controls was indicated. Validation in a silica-induced pulmonary fibrosis mouse model supported SPARC and MT-CO2 as potential signature genes for the prediction of silicosis. These findings highlight a strong association between silicosis and cuproptosis. Among CRGs, LOX, SPARC, MOXD1, ALB, MT-CO2, and AOC2 emerged as pivotal players in the context of silicosis by modulating CD8 T cells, regulatory T cells, M0 macrophages, and neutrophils.

Retracted: Rehmannia Radix Extract Relieves Bleomycin-Induced Pulmonary Fibrosis in Mice via Transforming Growth Factor β1 (TGF-β1).

The authors have requested retraction due to the identification of errors in the data. Reference: Xiaoming Hu, Dongzhe Zhu. Rehmannia Radix Extract Relieves Bleomycin-Induced Pulmonary Fibrosis in Mice via Transforming Growth Factor ß1 (TGF-ß1). Med Sci Monit, 2020; 26: e927240. DOI: 10.12659/MSM.927240.

Repositioning of ezetimibe for the treatment of idiopathic pulmonary fibrosis.

We previously identified ezetimibe, an inhibitor of Niemann-Pick C1-like intracellular cholesterol transporter 1 and European Medicines Agency-approved lipid-lowering agent, as a potent autophagy activator. However, its efficacy against pulmonary fibrosis has not yet been evaluated. This study aimed to determine whether ezetimibe has therapeutic potential against idiopathic pulmonary fibrosis. Primary lung fibroblasts isolated from both humans and mice were employed for mechanistic in vitro experiments. mRNA sequencing of human lung fibroblasts and gene set enrichment analysis were performed to explore the therapeutic mechanism of ezetimibe. A bleomycin-induced pulmonary fibrosis mouse model was used to examine in vivo efficacy of the drug. Tandem fluorescent-tagged microtubule-associated protein 1 light chain 3 transgenic mice were used to measure autophagic flux. Finally, the medical records of patients with idiopathic pulmonary fibrosis from three different hospitals were reviewed retrospectively, and analyses on survival and lung function were conducted to determine the benefits of ezetimibe. Ezetimibe inhibited myofibroblast differentiation by restoring the mechanistic target of rapamycin complex 1-autophagy axis with fine control of intracellular cholesterol distribution. Serum response factor, a potential autophagic substrate, was identified as a primary downstream effector in this process. Similarly, ezetimibe ameliorated bleomycin-induced pulmonary fibrosis in mice by inhibiting mechanistic target of rapamycin complex 1 activity and increasing autophagic flux, as observed in mouse lung samples. Patients with idiopathic pulmonary fibrosis who regularly used ezetimibe showed decreased rates of all-cause mortality and lung function decline. Our study presents ezetimibe as a potential novel therapeutic for idiopathic pulmonary fibrosis.

New tricks for old drugs- praziquantel ameliorates bleomycin-induced pulmonary fibrosis in mice

BackgroundPulmonary fibrosis is a chronic progressive disease with complex pathogenesis, short median survival time, and high mortality. There are few effective drugs approved for pulmonary fibrosis treatment. This study aimed to evaluate the effect of praziquantel (PZQ) on bleomycin (BLM)-induced pulmonary fibrosis.MethodsIn this study, we investigated the role and mechanisms of PZQ in pulmonary fibrosis in a murine model induced by BLM. Parameters investigated included survival rate, lung histopathology, pulmonary collagen deposition, mRNA expression of key genes involved in pulmonary fibrosis pathogenesis, the activity of fibroblast, and M2/M1 macrophage ratio.ResultsWe found that PZQ improved the survival rate of mice and reduced the body weight loss induced by BLM. Histological examination showed that PZQ significantly inhibited the infiltration of inflammatory cells, collagen deposition, and hydroxyproline content in BLM-induced mice. Besides, PZQ reduced the expression of TGF-β and MMP-12 in vivo and inhibited the proliferation of fibroblast induced by TGF-β in vitro. Furthermore, PZQ affected the balance of M2/M1 macrophages.ConclusionsOur study demonstrated that PZQ could ameliorate BLM-induced pulmonary fibrosis in mice by affecting the balance of M2/M1 macrophages and suppressing the expression of TGF-β and MMP-12. These findings suggest that PZQ may act as an effective anti-fibrotic agent for preventing the progression of pulmonary fibrosis.

Pirfenidone and nintedanib attenuate pulmonary fibrosis in mice by inhibiting the expression of JAK2.

Pirfenidone and nintedanib were approved by the Food and Drug Administration (FDA) for the treatment of idiopathic pulmonary fibrosis (IPF). These two drugs can slow the progression of the disease, but the specific mechanisms are not fully understood. In the current study, bleomycin (BLM) induced pulmonary fibrosis in mice was accompanied by high p-JAK2 expression in lung tissue, mainly in the nucleus. The expression of p-JAK2 significantly decreased after intragastric administration of pirfenidone and nintedanib. p-JAK2 is reportedly expressed mainly in the cytoplasm and exerts its effect by activating downstream p-STAT3 in the nucleus. In vivo experiments, pulmonary fibrosis was induced in mice with BLM and then treated with pirfenidone and nintedanib. The levels of transforming growth factor-β (TGF-β1), SP-A, SP-D and KL-6 in serum were measured by enzyme-linked immunosorbent assay (ELISA). Pathological staining was performed to assess lung fibrosis in mice, Western blot was performed to detect the expression levels of relevant proteins, and immunofluorescence was performed to observe the fluorescence expression of p-JAK2. In cellular experiments, MLE12 was stimulated with TGF-β1 and intervened with TGF-β1 receptor inhibitor and si-JAK2, pirfenidone and nintedanib, respectively, and the related protein expression levels were detected by Western blot. In both in vivo and in vitro experiments, pirfenidone and nintedanib were found to attenuate the expression of lung fibrosis markers by inhibiting the expression of JAK2, which may reduce the entry of p-JAK2 into the nucleus by downregulating JAK2 phosphorylation through inhibition of the TGF-β receptor. In contrast, inhibition of JAK2 expression greatly reduced the expression of TGF-β receptor and α-smooth muscles actin (a myofibroblast activation marker). In both in vivo and in vitro experiments, the present study demonstrated that TGF-β1 promotes JAK2 phosphorylation through a non-classical pathway, and conversely, inhibition of JAK2 expression affects the TGF-β1 signalling pathway. Therefore, we speculate that TGF-β1 and JAK2 signaling pathways interact with each other and participate in fibrosis.

Inhibition of sphingosine 1-phosphate receptor 3 ameliorates bleomycin-induced pulmonary fibrosis by suppressing macrophage M2 polarization

Pulmonary fibrosis is a devastating lung disease without effective treatment options. Sphingosine-1-phosphate receptor 3 (S1pr3), a receptor for the lipid signaling molecule sphingosine-1-phosphate, has been shown to mediate the development of pulmonary fibrosis, although the underlying mechanism is not fully understood. Here, we found increased expression of S1pr3 in the lung during the process of bleomycin-induced pulmonary fibrosis in mice and specific overexpression of S1pr3 in the infiltrated M2 macrophages. We constructed LysM-Cre+/S1pr3flox/flox mice, in which S1pr3 was conditionally depleted in myeloid cells, and this depletion protected mice from bleomycin-induced lung injury and fibrosis, with reduced M2 macrophage accumulation in the lung. Increased S1pr3 expression was found in bone marrow-derived macrophages after alternatively activated by IL4 ex vivo, while loss of S1pr3 attenuated IL-4-induced M2 polarization in bone marrow-derived macrophages by repressing the PI3K/Akt-Stat3 signaling pathway. Moreover, the S1pr3 inhibitors CAY10444 and TY52156 exerted protective effects on pulmonary fibrosis in mice. Taken together, our research showed that inhibition of S1pr3 ameliorates bleomycin-induced pulmonary fibrosis by reducing macrophage M2 polarization via the PI3K/Akt-Stat3 signaling pathway, indicating that S1pr3 may be a potential target for pulmonary fibrosis treatment.

8-Oxoguanine DNA glycosylase protects cells from senescence via the p53-p21 pathway.

Cellular senescence is an important factor leading to pulmonary fibrosis. Deficiency of 8-oxoguanine DNA glycosylase (OGG1) in mice leads to alleviation of bleomycin (BLM)-induced mouse pulmonary fibrosis, and inhibition of the OGG1 enzyme reduces the epithelial mesenchymal transition (EMT) in lung cells. In the present study, we find decreased expression of OGG1 in aged mice and BLM-induced cell senescence. In addition, a decrease in OGG1 expression results in cell senescence, such as increases in the percentage of SA-β-gal-positive cells, and in the p21 and p-H2AX protein levels in response to BLM in lung cells. Furthermore, OGG1 promotes cell transformation in A549 cells in the presence of BLM. We also find that OGG1 siRNA impedes cell cycle progression and inhibits the levels of telomerase reverse transcriptase (TERT) and LaminB1 in BLM-treated lung cells. The increase in OGG1 expression results in the opposite phenomenon. The mRNA levels of senescence-associated secretory phenotype (SASP) components, including IL-1α, IL-1β, IL-6, IL-8, CXCL1/CXCL2, and MMP-3, in the absence of OGG1 are obviously increased in A549 cells treated with BLM. Interestingly, we demonstrate that OGG1 binds to p53 to inhibit the activation of p53 and that silencing of p53 reverses the inhibition of OGG1 on senescence in lung cells. Additionally, the augmented cell senescence is shown in vivo in OGG1-deficient mice. Overall, we provide direct evidence in vivo and in vitro that OGG1 plays an important role in protecting tissue cells against aging associated with the p53 pathway.

Platycodin D improves pulmonary fibrosis in mice by down-regulating TRPC6 expression and reducing ROS production in lung fibroblasts

To assess the effect of platycodin D (PD) for alleviating pulmonary fibrosis in mice and explore the underlying mechanism. C57BL/6J mouse models of pulmonary fibrosis induced by bleomycin injection into the airway were treated with daily intragastric administration of 10 mg/kg PD for 28 days. The changes of pulmonary fibrosis and the expression and distribution of transient receptor potential cation channel subfamily C member 6 (TRPC6) were evaluated with immunohistochemistry, HE staining and Sirius Red staining. Western blotting was used to detect α-SMA expression in the lung tissues of the mice. Primary cultures of mouse lung fibroblasts were pretreated with PD (2.5, 5.0, and 10 μmol/L) or larixyl acetate (LA; 10 μmol/L) before exposure to 10 ng/mL transforming growth factor-β1 (TGF-β1), and the changes in cell survival rate, expressions of collagen Ⅰ, α-SMA and TRPC6, reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell proliferation capacity were assessed. Network pharmacology analysis was performed to explore the mechanism by which PD alleviated pulmonary fibrosis. PD treatment significantly alleviated pulmonary fibrosis and reduced α-SMA expression in BLM-induced mouse models (P<0.05). In TGF-β1-induced primary mouse lung fibroblasts, PD effectively inhibited the cell proliferation, reduced ROS production (P<0.0001), rescued the reduction of mitochondrial membrane potential (P<0.001), and inhibited the expressions of α-SMA and collagen Ⅰ (P<0.05). Network pharmacology analysis suggested that TRPC6 mediated the effect of PD for alleviating pulmonary fibrosis. Immunohistochemistry showed that PD significantly reduced TRPC6 expression in the lung tissues of BLM-induced mice. In primary mouse lung fibroblasts, PD significantly inhibited TGF-β1-induced TRPC6 expression (P<0.05), and LA treatment obviously lowered the expression levels of TRPC6, α-SMA and collagenⅠ (P<0.05). PD alleviated pulmonary fibrosis in mice possibly by down-regulating TRPC6 and reducing ROS production.

Research of miR-29a on TGF-β1/Smad3 pathway in pulmonary fibrosis induced by neodymium oxide

Objective: To exploring the regulatory effect of miR-29a on the transforming growth factor-β1 (TGF-β1) /Smad homolog 3 (Smad3) pathway during the process of rare earth neodymium oxide (Nd(2)O(3)) induced pulmonary fibrosis in mice. Methods: In March 2021, 72 SPF grade C57/BL6J male mice were selected and randomly divided into a control group, Nd(2)O(3) group, Nd(2)O(3)+miR-29a agomir group, and Nd(2)O(3)+NC agomir group, with 18 mice in each group. The Nd(2)O(3) group, Nd(2)O(3)+miR-29a agomir group, and Nd(2)O(3)+NC agomir group were treated with non exposed tracheal instillation, with a dust concentration of 250 mg/ml and a dust volume of 0.1 ml. The control group was given the same volume of physiological saline. After exposure to Nd(2)O(3), 0.1 ml (5 nmol) of miR-29a agomir was injected into the tail vein of mice in the Nd(2)O(3)+miR-29a agomir group every 3 days, while 0.1 ml of NC agomir was injected into the tail vein of mice in the Nd(2)O(3)+NC agomir group. On the 7 th, 14 th, and 28 th days after dust exposure, 6 mice were killed in each group, and the lung tissue of the mice was taken out. HE staining was used to observe the pathological status of the mouse lung tissue; ELISA method was used to detect the levels of TGF-β1 and connective tissue growth factor (CTGF) in lung tissue; Use qRT-PCR detection method to detect the expression level of TGF-β1 mRNA; Using immunofluorescence assay to detect the expression level of Smad3 in mouse lung tissue; Use bioinformatics websites such as TargetScan7 and miRDB to predict the target gene of miR-29a. When the metrological date were satisfied with normal distribution, Mean±SD was used for comparison between groups, t test was used for two indepent samples, and LSD method was used when the variance was homogeneity in pairwise comparison. Results: HE staining showed that the Nd(2)O(3) group of mice showed obvious infiltration of inflammatory cells and structural disorder of alveoli in the early stage of lung tissue. At 28 days, the collagen fibers in the mouse lung tissue increased and the lung tissue showed fibrotic honeycomb like changes. The degree of pulmonary fibrosis in the Nd(2)O(3)+miR-29a agomir group of mice was significantly reduced; The content of TGF-β1 and CTGF in the lung tissue of mice in the Nd(2)O(3)+miR-29a agomir group was lower than that in the Nd(2)O(3)+NC agomir group (P<0.05) ; The relative expression level of TGF-β1 in the lung tissue of mice in the Nd(2)O(3)+miR-29a agomir group was lower than that in the Nd(2)O(3)+NC agomir group (P<0.05) ; The expression level of Smad3 in the nucleus of the Nd(2)O(3)+miR-29a agomir group was lower than that of the Nd(2)O(3)+NC agomir group (P<0.05). The prediction results of bioinformatics websites have found 152 downstream target genes related to miR-29a, among which FBN1, MAP2K6, KPNB1, COL1A2, SNIP1, LAMC1, and SP1 genes may be related to the regulatory effect of miR-29a on TGF-β1/Smad3 signaling pathway. Conclusion: miR-29a may affect lung fibrosis induced by rare earth Nd(2)O(3) exposure in mice by regulating TGF-β1/Smad3 signaling pathway. Overexpression of miR-29a may inhibit TGF-β1/Smad3 signaling pathway and reduce the degree of pulmonary fibrosis in mice.

The SIRT1/Nrf2 signaling pathway mediates the anti-pulmonary fibrosis effect of liquiritigenin

BackgroundAt present, the treatment options available for idiopathic pulmonary fibrosis are both limited and often come with severe side effects, emphasizing the pressing requirement for innovative therapeutic alternatives. Myofibroblasts, which hold a central role in pulmonary fibrosis, have a close association with the Smad signaling pathway induced by transforming growth factor-β1 (TGF-β1) and the transformation of myofibroblasts driven by oxidative stress. Liquiritigenin, an active compound extracted from the traditional Chinese herb licorice, boasts a wide array of biomedical properties, such as anti-fibrosis and anti-oxidation. The primary objective of this study was to examine the impact of liquiritigenin on bleomycin-induced pulmonary fibrosis in mice and the underlying mechanisms.MethodsThe anti-pulmonary fibrosis and anti-oxidant effects of liquiritigenin in vivo were tested by HE staining, Masson staining, DHE staining and bio-chemical methods. In vitro, primary mouse lung fibroblasts were treated with TGF-β1 with or without liquiritigenin, the effects of liquiritigenin in inhibiting differentiation of myofibroblasts and facilitating the translocation of Nrf2 were valued using Quantitative real-time polymerase chain reaction (Q-PCR), western blotting and immunofluorescence. Nrf2 siRNA and SIRT1 siRNA were used to investigate the mechanism underlies liquiritigenin’s effect in inhibiting myofibroblast differentiation.ResultsLiquiritigenin displayed a dose-dependent reduction effect in bleomycin-induced fibrosis. In laboratory experiments, it was evident that liquiritigenin possessed the ability to enhance and activate sirtuin1 (SIRT1), thereby facilitating the nuclear translocation of Nrf2 and mitigating the oxidative stress-induced differentiation of primary mouse myofibroblasts. Moreover, our investigation unveiled that SIRT1 not only regulated myofibroblast differentiation via Nrf2-mediated antioxidant responses against oxidative stress but also revealed liquiritigenin's activation of SIRT1, enabling direct binding to Smad. This led to decreased phosphorylation of the Smad complex, constrained nuclear translocation, and suppressed acetylation of the Smad complex, ultimately curtailing the transcription of fibrotic factors. Validation in live subjects provided substantial evidence for the anti-fibrotic efficacy of liquiritigenin through the SIRT1/Nrf2 signaling pathway.ConclusionsOur findings imply that targeting myofibroblast differentiation via the SIRT1/Nrf2 signaling pathway may constitute a pivotal strategy for liquiritigenin-based therapy against pulmonary fibrosis.

Pathogenic Characteristics of an Infection with Canine Influenza Virus and Streptococcus equi subsp. zooepidemicus Alone or in Combination in Mice

Both Streptococcus equi subsp. zooepidemicus (SEZ) and canine influenza virus (CIV) are two important pathogens causing infectious respiratory disease in dogs and are frequently codetected in respiratory secretions. However, the clinical significance of viral/bacterial coinfection remains unknown. This study investigated the pathogenic characteristics of infection with CIV and SEZ alone or in combination in mice. Our data indicated that the severity of the disease is related to the challenge order of CIV and SEZ. Coinfection of CIV and SEZ induced higher weight loss in mice than single infection, except for the VB group (viral followed by secondary bacterial infection). Compared with the concurrent or sequential infection groups of CIV and SEZ, mice in the CIV–SEZ preincubation group exhibited more obvious weight loss, higher mortality, and significantly enhanced burden of SEZ and CIV in tissues. Interestingly, viral and bacterial preincubation before coinfection caused typical pulmonary fibrosis in mice. Correspondingly, transforming growth factor (TGF)-β was upregulated, and its canonical small mother against decapentaplegic (Smad) 2/3 signaling was noticeably induced. Further investigation indicated that the activity of the viral neuraminidase (NA) enzyme upon sialic acid was considerably increased due to the direct interaction of CIV with SEZ, which may be related to the activation of the TGF-β signaling pathway. These findings implicate an unexpected contribution of the direct interaction between CIV and SEZ to synergistic pathogenicity.

Integrating network pharmacology, experimental validation and molecular docking to reveal the alleviation of Yinhuang granule on idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the abnormal proliferation of fibroblast and excessive deposition of extracellular matrix (ECM), accompanied by inflammation and ultimately respiratory failure. Yinhuang granule (YHG), with clinical properties of clearing heat, detoxifying and anti-inflammation, is commonly used to heal upper respiratory diseases in China for decades. PurposeTo explore the improvement of YHG on bleomycin (BLM)-induced IPF in mice and its possible engaged mechanism. MethodsThe mortality rate was recorded, lung function was determined and hematoxylin-eosin (H&E) staining was carried out to explore the alleviation of YHG on BLM-caused IPF in mice. Hydroxyproline, collagen I and collagen III contents were detected, and Sirius red and Masson staining were conducted to evaluate YHG's alleviation on lung fibrosis. The underlying mechanism was predicted by network pharmacology, and confirmed by Real-time polymerase chain reaction (RT-PCR), Western-blot (WB) and enzyme linked immunosorbent assay (ELISA). The binding affinity between related key proteins and active compounds in YHG was calculated by using molecular docking, and further validated by cellular thermal shift assay (CESTA). ResultsYHG (400, 800 mg/kg) weakened lung damage and pulmonary fibrosis in mice induced by BLM. Network pharmacology and experimental validation displayed that inflammation and angiogenesis participated in the YHG-provided improvement on IPF, and key involved molecules included tumor necrosis factor-α (TNFα), vascular endothelial growth factor-A (VEGFA), interleukine-6 (IL-6), etc. The data of molecular docking presented that some main active compounds from YHG had a high binding affinity with TNFR1 or VEGFR2, and some of them were further validated by CESTA. ConclusionYHG effectively improved the BLM-induced IPF in mice via reducing inflammation and angiogenesis.

Mangiferin alleviates diabetic pulmonary fibrosis in mice via inhibiting endothelial-mesenchymal transition through AMPK/FoxO3/SIRT3 axis.

Diabetes mellitus results in numerous complications. Diabetic pulmonary fibrosis (DPF), a late pulmonary complication of diabetes, has not attracted as much attention as diabetic nephropathy and cardiomyopathy. Mangiferin (MF) is a natural small molecular compound that exhibits a variety of pharmacological effects including anti-inflammatory, anti-cancer, anti-diabetes, and anti-fibrosis effects. In this study, we investigated whether long-term diabetes shock induces DPF, and explored whether MF had a protective effect against DPF. We first examined the lung tissues and sections of 20 diabetic patients obtained from discarded lung surgical resection specimens and found that pulmonary fibrosis mainly accumulated around the pulmonary vessels, accompanied by significantly enhanced endothelial-mesenchymal transition (EndMT). We established a mouse model of DPF by STZ injections. Ten days after the final STZ injection, the mice were administered MF (20, 60 mg/kg, i.g.) every 3 days for 4 weeks, and kept feeding until 16 weeks and euthanized. We showed that pulmonary fibrotic lesions were developed in the diabetic mice, which began around the pulmonary vessels, while MF administration did not affect long-term blood glucose levels, but dose-dependently alleviated diabetes-induced pulmonary fibrosis. In human umbilical vein endothelial cells (HUVECs), exposure to high glucose (33.3 mM) induced EndMT, which was dose-dependently inhibited by treatment with MF (10, 50 μM). Furthermore, MF treatment promoted SIRT3 expression in high glucose-exposed HUVECs by directly binding to AMPK to enhance the activity of FoxO3, which finally reversed diabetes-induced EndMT. We conclude that MF attenuates DPF by inhibiting EndMT through the AMPK/FoxO3/SIRT3 axis. MF could be a potential candidate for the early prevention and treatment of DPF.

Alpinetin ameliorates bleomycin-induced pulmonary fibrosis by repressing fibroblast differentiation and proliferation

ObjectiveIdiopathic pulmonary fibrosis (IPF) is a progressive and irreversible interstitial lung disease with a poor prognosis. Alpinetin (ALP), derived from Alpinia katsumadai Hayata, has shown potential as a therapeutic measure of various diseases. However, the utilization of ALP in managing pulmonary fibrosis and its underlying mechanisms are still not fully understood. MethodsA well-established mouse model of pulmonary fibrosis induced by bleomycin (BLM) was used in this study. The antifibrotic effects of ALP on histopathologic manifestations and expression levels of fibrotic markers were examined. Subsequently, the impact of ALP on fibroblast differentiation, proliferation, apoptosis, and associated signaling pathways was investigated to elucidate the underlying mechanisms. ResultsIn the present study, we observed that ALP effectively mitigated BLM-induced pulmonary fibrosis in mice, as evidenced by histopathological manifestations and the expression levels of fibrotic markers. Furthermore, the in vitro experiments demonstrated that ALP treatment attenuated the ability of fibroblasts to differentiate into myofibroblasts. Mechanically, our findings provided evidence that ALP suppressed fibroblast-to-myofibroblast differentiation by repressing TGF-β/ALK5/Smad signaling pathway. ALP was found to possess the capability of inhibiting fibroblast proliferation and promoting apoptosis of fibroblasts induced by TGF-β. ConclusionIn general, ALP may exert therapeutic effects on pulmonary fibrosis by modulating the differentiation, proliferation, and apoptosis of fibroblasts. Although its safety has been demonstrated in mice, further studies are required to investigate the efficacy of ALP in treatment of patients with IPF.

Leech extract alleviates idiopathic pulmonary fibrosis by TGF-β1/Smad3 signaling pathway

Ethnopharmacological relevanceLeech, as a traditional Chinese medicine for the treatment of blood circulation and blood stasis, was also widely used to cure pulmonary fibrosis in China. In clinical practice, some traditional Chinese medicine preparation such as Shui Zhi Xuan Bi Hua Xian Tang and Shui Zhi Tong Luo Capsule composed of leech, could improve the clinical symptoms and pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). However, the material basis of the leech in the treatment of IPF were not yet clear. Aim of the studyScreen out the components of leech that have the anti-pulmonary fibrosis effects, and further explore the therapeutic mechanism of the active components. Materials and methodsIn this study, the different molecular weight components of leech extract samples were prepared using the semi-permeable membranes with different pore sizes. The therapeutic effects of the leech extract groups with molecular weight greater than 10 KDa (>10 KDa group), between 3 KDa and 10 KDa (3–10 KDa group), and less than 3 KDa (<3 KDa group) on pulmonary fibrosis were firstly investigated by cell proliferation and cytotoxicity assay (MTT), cell wound healing assay, immunofluorescence staining (IF) and Western blot (WB) assay through the TGF-β1-induced fibroblast cell model. Then bleomycin-induced pulmonary fibrosis (BML-induced PF) mouse model was constructed to investigate the pharmacological activities of the active component group of leech extract in vivo. Pathological changes of the mouse lung were observed by hematoxylin-eosin staining (H&E) and Masson's trichrome staining (Masson). The hydroxyproline (HYP) content of lung tissues was quantified by HYP detection kit. The levels of extracellular matrix-related fibronectin (FN) and collagen type Ⅰ (Collagen Ⅰ), pyruvate kinase M2 (PKM2) monomer and Smad7 protein were determined via WB method. PKM2 and Smad7 protein were further characterized by IF assays. ResultsUsing TGF-β1-induced HFL1 cell line as a PF cell model, the in vitro results demonstrated that the >10 KDa group could significantly inhibited the cell proliferation and migration, downregulated the expression level of cytoskeletal protein vimentin and α-smooth muscle actin (α-SMA), and reduced the deposition of FN and Collagen Ⅰ. In the BML-induced PF mouse model, the >10 KDa group significantly reduced the content of HYP, downregulated the expression levels of FN and Collagen Ⅰ in lung tissues, and delayed the pathological changes of lung tissue structure. The results of WB and IF assays further indicated that the >10 KDa group could up-regulate the expression level of PKM2 monomer and Smad7 protein in the cellular level, thereby delaying the progression of pulmonary fibrosis. ConclusionsOur study revealed that the >10 KDa group was the main material basis of the leech extract that inhibited pulmonary fibrosis through TGF-β1/Smad3 signaling pathway.

Retracted: Influence of miRNA-30a-5p on Pulmonary Fibrosis in Mice with Streptococcus pneumoniae Infection through Regulation of Autophagy by Beclin-1.

[This retracts the article DOI: 10.1155/2021/9963700.].

Unraveling the mechanism of ethyl acetate extract from Prismatomeris connata Y. Z. Ruan root in treating pulmonary fibrosis: insights from bioinformatics, network pharmacology, and experimental validation.

Pulmonary fibrosis is a terminal lung disease characterized by fibroblast proliferation, extracellular matrix accumulation, inflammatory damage, and tissue structure destruction. The pathogenesis of this disease, particularly idiopathic pulmonary fibrosis (IPF), remains unknown. Macrophages play major roles in organ fibrosis diseases, including pulmonary fibrosis. The phenotype and polarization of macrophages are closely associated with pulmonary fibrosis. A new direction in research on anti-pulmonary fibrosis is focused on developing drugs that maintain the stability of the pulmonary microenvironment. We obtained gene sequencing data and clinical information for patients with IPF from the GEO datasets GSE110147, GSE15197, GSE24988, GSE31934, GSE32537, GSE35145, GSE53845, GSE49072, GSE70864, and GSE90010. We performed GO, KEGG enrichment analysis and GSEA analysis, and conducted weighted gene co-expression network analysis. In addition, we performed proteomic analysis of mouse lung tissue. To verify the results of bioinformatics analysis and proteomic analysis, mice were induced by intratracheal instillation of bleomycin (BLM), and gavaged for 14 days after modeling. Respiratory function of mice in different groups was measured. Lungtissues were retained for histopathological examination, Western Blot and real-time quantitative PCR, etc. In addition, lipopolysaccharide, interferon-γ and interleukin-4 were used to induce RAW264.7 cells for 12h in vitro to establish macrophage inflammation and polarization model. At the same time, HG2 intervention was given. The phenotype transformation and cytokine secretion of macrophages were investigated by Western Blot, RT-qPCR and flow cytometry, etc. Through bioinformatics analysis and experiments involving bleomycin-induced pulmonary fibrosis in mice, we confirmed the importance of macrophage polarization in IPF. The analysis revealed that macrophage polarization in IPF involves a change in the phenotypic spectrum. Furthermore, experiments demonstrated high expression of M2-type macrophage-associated biomarkers and inducible nitric oxide synthase, thus indicating an imbalance in M1/M2 polarization of pulmonary macrophages in mice with pulmonary fibrosis. Our investigation revealed that the ethyl acetate extract (HG2) obtained from the roots of Prismatomeris connata Y. Z. Ruan exhibits therapeutic efficacy against bleomycin-induced pulmonary fibrosis. HG2 modulates macrophage polarization, alterations in the TGF-β/Smad pathway, and downstream protein expression in the context of pulmonary fibrosis. On the basis of our findings, we believe that HG2 has potential as a novel traditional Chinese medicine component for treating pulmonary fibrosis.

Toosendanin Restrains Idiopathic Pulmonary Fibrosis by Inhibiting ZEB1/CTBP1 Interaction.

Extensive deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) is due to hyperactivation and proliferation of pulmonary fibroblasts. However, the exact mechanism is not clear. This study focused on the role of CTBP1 in lung fibroblast function, elaborated its regulation mechanism, and analyzed the relationship between CTBP1 and ZEB1. Meanwhile, the antipulmonary fibrosis effect and its molecular mechanism of Toosendanin were studied. Human IPF fibroblast cell lines (LL-97A and LL-29) and normal fibroblast cell lines (LL-24) were cultured in vitro. The cells were stimulated with FCS, PDGF-BB, IGF-1, and TGF-β1, respectively. BrdU detected cell proliferation. The mRNA expression of CTBP1 and ZEB1 was detected by QRT-PCR. Western blotting was used to detect the expression of COL1A1, COL3A1, LN, FN, and α-SMA proteins. An animal model of pulmonary fibrosis was established to analyze the effects of CTBP1 silencing on pulmonary fibrosis and lung function in mice. CTBP1 was up-regulated in IPF lung fibroblasts. Silencing CTBP1 inhibits growth factor-driven proliferation and activation of lung fibroblasts. Overexpression of CTBP1 promotes growth factor-driven proliferation and activation of lung fibroblasts. Silencing CTBP1 reduced the degree of pulmonary fibrosis in mice with pulmonary fibrosis. Western blot, CO-IP, and BrdU assays confirmed that CTBP1 interacts with ZEB1 and promotes the activation of lung fibroblasts. Toosendanin can inhibit the ZEB1/CTBP1protein interaction and further inhibit the progression of pulmonary fibrosis. CTBP1 can promote the activation and proliferation of lung fibroblasts through ZEB1. CTBP1 promotes lung fibroblast activation through ZEB1, thereby increasing excessive deposition of ECM and aggravating IPF. Toosendanin may be a potential treatment for pulmonary fibrosis. The results of this study provide a new basis for clarifying the molecular mechanism of pulmonary fibrosis and developing new therapeutic targets.

Metformin enhanced the effect of pirfenidone on pulmonary fibrosis in mice.

The aim of the study is to observe the anti-inflammatory and antioxidative stress effects of metformin on bleomycin (BLM)-induced pulmonary fibrosis in mice. Mice with BLM-induced pulmonary fibrosis were treated with pirfenidone, metformin, pirfenidone plus metformin and the NADPH oxidase 4 (NOX4) inhibitor diphenyleneiodonium chloride (DPI). Pathological changes and hydroxyproline (HPO) levels were examined in the lung tissue of mice with pulmonary fibrosis. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) levels in lung tissue were determined. Compared with pirfenidone, pirfenidone plus metformin could reduce alveolar damage and collagen fibre deposition and alleviate BLM-induced pulmonary fibrosis. Lung HPO levels were significantly lower in the PFD + MET group than in the BLM group (p < 0.05). SOD levels in the lungs of mice were increased in the PFD + MET group than in the BLM group (p < 0.05). Metformin and pirfenidone plus metformin can reduce MDA levels (p < 0.05). Pirfenidone plus metformin could reduce HPO levels, increase SOD levels, and reduce MDA levels in the lungs of mice. There was a significant correlation between the HPO level and the Ashcroft score (r = 0.520, p < 0.001). Metformin enhanced the antifibrotic effects of pirfenidone on BLM-treated mice. Moreover, these findings provide an experimental basis for examining whether metformin can improve the antifibrotic effects of pirfenidone on patients with idiopathic pulmonary fibrosis (IPF). It has broad therapeutic prospects for patients with IPF.