Paraquat-induced Pulmonary Fibrosis Research Articles

Mechanism of action of miR-15a-5p and miR-152-3p in paraquat-induced pulmonary fibrosis through Wnt/β-catenin signaling mediation

Background miRNAs are small, conserved, single-stranded non-coding RNA that are typically transported by exosomes for their functional roles. The therapeutic potential of exosomal miRNAs has been explored in various diseases including breast cancer, pancreatic cancer, cholangiocarcinoma, skin diseases, Alzheimer’s disease, stroke, and glioma. Pathophysiological processes such as cellular inflammation, apoptosis, necrosis, immune dysfunction, and oxidative stress are closely associated with miRNAs. Internal and external factors such as tissue ischemia, hypoxia, pathogen infection, and endotoxin exposure can trigger these reactions and are linked to miRNAs. Paraquat-induced fibrosis is a protracted process that may not manifest immediately after injury but develops during bodily recovery, providing insights into potential miRNA intervention treatments. Rationale These findings could potentially be applied for further pharmaceutical research and clinical therapy of paraquat-induced pulmonary fibrosis, and are likely to be of great interest to clinicians involved in lung fibrosis research. Methodology Through a literature review, we identified an association between miR-15a-5p and miR-152-3p and their involvement in the Wnt signaling pathway. This allowed us to deduce the molecular mechanisms underlying regulatory interactions involved in paraquat-induced lung fibrosis. Results miR-15a-5p and miR-152-3p play roles in body repair processes, and pulmonary fibrosis can be considered a form of reparative response by the body. Although the initial purpose of fibrotic repair is to restore normal body function, excessive tissue fibrosis, unlike scar formation following external skin trauma, can significantly and adversely affect the body. Modulating the Wnt/β-catenin signaling pathway is beneficial in alleviating tissue fibrosis in various diseases. Conclusions In this study, we delineate the association between miR-15a-5p and miR-152-3p and the Wnt/β-catenin signaling pathway, presenting a novel concept for addressing paraquat-induced pulmonary fibrosis.

Early diagnosis and staging of paraquat-induced pulmonary fibrosis using [18F]F-FAPI-42 PET/CT imaging

BackgroundParaquat (PQ) -induced pulmonary fibrosis poses a significant medical challenge due to limited treatment options and high mortality rates. Consequently, there is an urgent need for early diagnosis and accurate staging to facilitate appropriate treatment strategies. In this study, we assessed the diagnostic potential of [18F]F-FAPI-42 PET/CT imaging for early detection and disease staging in a rat model of PQ-induced lung fibrosis.MethodsAfter administering 80 mg/kg of PQ orally to Sprague-Dawley rats, we intravenously injected 3-3.5 MBq of [18F]F-FAPI-42 on day 7, 14, and 21 post-dosing. Dynamic PET/CT imaging was carried out for one hour immediately after the administration of [18F]F-FAPI-42. Subsequently, the lung tissues were collected for Hematoxylin and Eosin (HE) staining, Masson’s trichrome staining, and NOTA-FAPI-04-MB fluorescent probe staining. Data analysis was performed using the Imalytics preclinical software, and the mean standardized uptake value (SUVmean) was calculated.ResultsPET signals revealed that in areas with evident lesions on CT, the SUVmean on day 14 was significantly higher than on day 7 and 21, indicating that changes in fibrosis activity levels contribute to the staging of pulmonary fibrosis. Additionally, the NOTA-FAPI-04-MB fluorescent probe staining also demonstrated the most pronounced probe uptake on day 14. In regions without apparent lesions on CT, the SUVmean gradually increased from day 7 to day 21, reflecting ongoing fibrotic activity. Moreover, HE staining and Masson’s trichrome staining did not reveal pulmonary fibrosis, while PET imaging was able to detect it, serving the purpose of early diagnosis. At 30 min and 60 min, the target-to-background ratio (TBR) of the PQ groups on day 7, 14, and 21 was significantly higher than the control group, suggesting a high specificity of [18F]F-FAPI-42 binding to activated fibroblasts.Conclusion[18F]F-FAPI-42 PET/CT imaging enables early diagnosis and staging of PQ-induced pulmonary fibrosis, demonstrating its feasibility and potential for characterizing early disease stages.

Inhibition of SMYD2 attenuates paraquat-induced pulmonary fibrosis by inhibiting the epithelial-mesenchymal transition through the GLIPR2/ERK/p38 axis

Paraquat (PQ) poisoning leads to irreversible fibrosis in the lungs with high mortality and no known antidote. In this study, we investigated the effect of the SET and MYND domain containing 2 (SMYD2) on PQ-induced pulmonary fibrosis (PF) and its potential mechanisms. We established an in vivo PQ-induced PF mouse model by intraperitoneal injection of PQ (20 mg/kg) and in vitro PQ (25 μM)-injured MLE-12 cell model. On the 15th day of administration, tissue injury, inflammation, and fibrosis in mice were evaluated using various methods including routine blood counts, blood biochemistry, blood gas analysis, western blotting, H&E staining, ELISA, Masson staining, and immunofluorescence. The findings indicated that AZ505 administration mitigated tissue damage, inflammation, and collagen deposition in PQ-poisoned mice. Mechanistically, both in vivo and in vitro experiments revealed that AZ505 treatment suppressed the PQ-induced epithelial-mesenchymal transition (EMT) process by downregulating GLI pathogenesis related 2 (GLIPR2) and ERK/p38 pathway. Further investigations demonstrated that SMYD2 inhibition decreased GLIPR2 methylation and facilitated GLIPR2 ubiquitination, leading to GLIPR2 destabilization in PQ-exposed MLE-12 cells. Moreover, rescue experiments conducted in vitro demonstrated that GLIPR2 overexpression eliminated the inhibitory effect of AZ505 on the ERK/p38 pathway and EMT. Our results reveal that the SMYD2 inhibitor AZ505 may act as a novel therapeutic candidate to suppress the EMT process by modulating the GLIPR2/ERK/p38 axis in PQ-induced PF.

Exosomes from senescent epithelial cells activate pulmonary fibroblasts via the miR-217-5p/Sirt1 axis in paraquat-induced pulmonary fibrosis

BackgroundParaquat (PQ) is a widely used and highly toxic herbicide that poses a significant risk to human health. The main consequence of PQ poisoning is pulmonary fibrosis, which can result in respiratory failure and potentially death. Our research aims to uncover a crucial mechanism in which PQ poisoning induces senescence in epithelial cells, ultimately regulating the activation of pulmonary fibroblasts through the exosomal pathway.MethodsCellular senescence was determined by immunohistochemistry and SA-β-Gal staining. The expression of miRNAs was measured by qPCR. Pulmonary fibroblasts treated with specific siRNA of SIRT1 or LV-SIRT1 were used to analysis senescent exosomes-mediated fibroblasts activation. Luciferase reporter assay and western blot were performed to elucidated the underlying molecular mechanisms. The effects of miR-217-5p antagomir on pulmonary fibrosis were assessed in PQ-poisoned mice models.ResultsImpairing the secretion of exosomes effectively mitigates the harmful effects of senescent epithelial cells on pulmonary fibroblasts, offering protection against PQ-induced pulmonary fibrosis in mice. Additionally, we have identified a remarkable elevation of miR-217-5p expression in the exosomes of PQ-treated epithelial cells, which specifically contributes to fibroblasts activation via targeted inhibition of SIRT1, a protein involved in cellular stress response. Remarkably, suppression of miR-217-5p effectively impaired senescent epithelial cells-induced fibroblasts activation. Further investigation has revealed that miR-217-5p attenuated SIRT1 expression and subsequently resulted in enhanced acetylation of β-catenin and Wnt signaling activation.ConclusionThese findings highlight a potential strategy for the treatment of pulmonary fibrosis induced by PQ poisoning. Disrupting the communication between senescent epithelial cells and pulmonary fibroblasts, particularly by targeting the miR-217-5p/SIRT1/β-catenin axis, may be able to alleviate the effects of PQ poisoning on the lungs.

Amphibian-derived peptide RL-RF10 ameliorates paraquat-induced pulmonary fibrosis injury

Pulmonary fibrosis is the result of dysfunctional repair after lung tissue injury, characterized by fibroblast proliferation and massive extracellular matrix aggregation. Once fibrotic lesions develop, effective treatment is difficult, with few drugs currently available. Here, we identified a short cyclic decapeptide RL-RF10 derived from frog skin secretions as a potential novel lead molecule for the amelioration of pulmonary fibrosis. In vivo experiments indicated that RL-RF10 treatment ameliorated lung histopathological damage and fibrogenesis after paraquat (PQ) induction in a concentration-dependent manner. On day 7, bronchoalveolar lavage fluid assays performed on mice showed that RL-RF10 exerted anti-inflammatory effects by decreasing the expression of inflammation-related factors, including transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α, in lung tissue. In addition, RL-RF10 down-regulated the levels of collagen I, collagen III, and vimentin, while increasing the expression of E-cadherin to inhibit epithelial-mesenchymal transition. Further research demonstrated that the SMAD2/3 signaling pathway, which is strongly linked to TGF-β1, played a critical function in enhancing the pulmonary fibrosis relief achieved by RL-RF10. Both in vivo and in vitro assays showed that RL-RF10 treatment led to a significant reduction in the phosphorylation levels of SMAD2 and SMAD3 following PQ induction. Overall, we investigated the protective effects and underlying mechanisms of the RL-RF10 peptide against pulmonary fibrosis and demonstrated its potential as a novel therapeutic drug candidate for the treatment of pulmonary fibrotic diseases.

The novel peptide DR4penA attenuates the bleomycin- and paraquat-induced pulmonary fibrosis by suppressing the TGF-β/Smad signaling pathway.

Pulmonary fibrosis (PF), which is caused by continuous alveolar epithelial cell injury and abnormal repair, is referred to as a difficult disease of the lung system by the World Health Organization due to its rapid progression, poor prognosis, and high mortality rate. However, there is still a lack of ideal therapeutic strategies. The peptide DR8 (DHNNPQIR-NH2 ), which is derived from rapeseed, exerted antifibrotic activity in the lung, liver, and kidney in our previous studies. By studying the structure-activity relationship and rational design, we introduced an unnatural hydrophobic amino acid (α-(4-pentenyl)-Ala) into DR8 and screened the novel peptide DR4penA (DHNα-(4-pentenyl)-APQIR-NH2 ), which had higher anti-PF activity, higher antioxidant activity and a longer half-life than DR8. Notably, DR4penA attenuated bleomycin- and paraquat-induced PF, and the anti-PF activity of DR4penA was equivalent to that of pirfenidone. Additionally, DR4penA suppressed the TGF-β/Smad pathway in TGF-β1-induced A549 cells and paraquat-induced rats. This study demonstrates that the novel peptide DR4penA is a potential candidate compound for PF therapy, and its antifibrotic activity in different preclinical models of PF provides a theoretical basis for further study.

2-Methoxyestradiol ameliorates paraquat-induced pulmonary fibrosis by inhibiting the TGF-β1/Smad2/3 signaling pathway

Paraquat (PQ) is a highly effective and highly toxic herbicide that is highly toxic to both humans and animals. Pulmonary fibrosis is the primary cause of fatality in patients with PQ poisoning, there is no effective drug treatment yet. 2-Methoxyestradiol (2ME) is a natural metabolite of estradiol with anti-tumor, anti-angiogenesis, and anti-proliferative effects. Whether 2ME has the potential to inhibit pulmonary fibrosis induced by PQ is unclear. This study aims to investigate the potential effects and mechanism of 2ME on PQ-induced pulmonary fibrosis. C57BL/6 mice and A549 cells were exposed to PQ to establish pulmonary fibrosis model. In vivo, Hematoxylin and eosin (H&E) staining was utilized to assess the pathological characteristics. Masson's trichrome staining was employed to evaluate the collagen deposition. Western blot and immunohistochemistry were conducted to determine the expressions of fibrosis markers. In vitro, the expressions of epithelial–mesenchymal transition (EMT) markers were detected using western blot and immunofluorescence to evaluated the potential inhibition of PQ-induced EMT by 2ME. And proteins associated with the TGF-β1/Smad2/3 signaling pathway were measured by western blot in vivo and in vitro. The result found that 2ME can ameliorated PQ-induced pulmonary fibrosis and inhibit the activation of TGF-β1/Smad2/3 signaling pathway. These findings suggest that 2ME may serve as a potential therapeutic agent for treating PQ-induced pulmonary fibrosis.

Chlorogenic acid protective effects on paraquat-induced pulmonary oxidative damage and fibrosis in rats.

Paraquat (PQ) is a widely used herbicide that can cause severe oxidative and fibrotic injuries in lung tissue. Due to the antioxidant and anti-inflammatory properties of chlorogenic acid (CGA), the present study investigated its effects on PQ-induced pulmonary toxicity. To this end, 30 male rats were randomly categorized into five groups of six. Initially, the first and third groups were treated intraperitoneally (IP) with normal saline and CGA (80 mg/kg) for 28 consecutive days, respectively. The second, fourth, and fifth groups were treated with normal saline and 20 and 80 mg/kg of CGA for 28 consecutive days, respectively, and received a single dose of PQ (IP, 20 mg/kg) on Day 7. Then, the animals were anesthetized with ketamine and xylazine, and lung tissue samples were collected for biochemical and histological examinations. The results showed that PQ significantly increased hydroxyproline (HP) and lipid peroxidation (LPO) and decreased the lung tissue antioxidant capacity. In addition, myeloperoxidase (MPO) activity increased significantly, while glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) activity declined substantially. The administration of therapeutic doses of CGA could prevent the oxidative, fibrotic, and inflammatory effects of PQ-induced lung toxicity, and these changes were consistent with histological observations. In conclusion, CGA may improve the antioxidant defense of lung tissue and prevent the spread of inflammation and the development of PQ-induced fibrotic injuries by enhancing antioxidant enzymes and preventing inflammatory cell infiltration.

Therapeutic potential of amitriptyline for paraquat-induced pulmonary fibrosis: Involvement of caveolin-1-mediated anti-epithelial-mesenchymal transition and inhibition of apoptosis

ObjectiveTreatment of pulmonary fibrosis caused by paraquat (PQ) poisoning remains problematic. Amitriptyline (AMT) has multiple pharmacological effects. Here we investigated the anti-fibrotic effect of AMT on PQ-induced pulmonary fibrosis and its possible mechanism. MethodsC57BL/6 mice were randomly divided into control, PQ, PQ + AMT and AMT groups. Histopathology of the lungs, blood gas analysis, and levels of hydroxyproline (HYP), transforming growth factor β1 (TGF-β1) and interleukin 17 (IL-17) were measured. The siRNA transfection inhibited caveolin-1 in A549 cells, which induced epithelial-mesenchymal transition (EMT) by PQ and followed intervention with AMT. E-cadherin, N-cadherin, α-smooth muscle actin (α-SMA) and caveolin-1 were studied by immunohistochemistry and western blot analysis. The apoptosis rate was measured by flow cytometry. ResultsCompared with the PQ group, the PQ + AMT group displayed mild pathological changes in pulmonary fibrosis, lower HYP, IL-17 and TGF- β1 levels in lung, but high TGF- β1 in serum. Levels of N-cadherin and α-SMA in the lungs were significantly decreased, but caveolin-1 was increased, while SaO2 and PaO2 levels were higher. Compared with the PQ group, the apoptosis rate, N-cadherin and α-SMA levels in A549 cells were significantly decreased after PQ treatment and high dose AMT intervention (p < 0.01). The expressions of E-cadherin, N-cadherin and α-SMA in the PQ-induced cells transfected with caveolin-1 siRNA or siControl RNA were significantly different (p < 0.01), but the apoptosis rate was unaltered. ConclusionAMT inhibited PQ-induced EMT in A549 cells and improved lung histopathology and oxygenation in mice by up-regulating caveolin-1.

Effect of pirfenidone on paraquat-induced pulmonary fibrosis in rats

Objective: To construct paraquat (PQ) poisoning rat model and to explore the effect of pirfenidone (PFD) on PQ-induced pulmonary fibrosis. Methods: In April 2017, male 6-8 week-old Wistar rats were selected, and PQ was administered intraperitoneally at one time. PFD was administered by gavage 2 hours after poisoning. The daily gavage doses were 100, 200 and 300 mg/kg, and the rats were divided into physiological saline group, PQ group, PQ+PFD 100 group, PQ+PFD 200 group, PQ+PFD 300 group, with 10 rats in each group at each observation time point. The pathological changes of lung tissue at different time points (the 1st, 3rd, 7th, 14th, 28th, 42nd and 56th days) after poisoning and the effect of PFD intervention with different dose on PQ-induced pulmonary fibrosis were observed. Pathological evaluation of lung tissue was performed by Ashcroft scale method. The PQ+PFD 200 group was selected to further explore the pathological changes of lung tissue, the contents of hydroxyproline and malondialdehyde in lung tissue were determined.And the tumor necrosis factor (TNF) -α, interleukin (IL) -6, transforming growth factor (TGF) -β1, fibroblast growth factor (FGF) -B, platelet-derived growth factor (PDGF) -AB, insulin-like growth factor (IGF) -1 and PQ concentrations in serum and lung tissue were determined. Results: On the 1st to 7th day after PQ exposure, rats developed lung inflammation, which was aggravated on the 7th to 14th day, and pulmonary fibrosis appeared on the 14th to 56th day. Compared with PQ group, the Ashcroft scores of lung fibrosis in PQ+PFD 200 group and PQ+PDF 300 group decreased significantly in 7th and 28th day (P<0.05), while the Ashcroft score of lung fibrosis in PQ+PFD 100 group had no significant difference (P>0.05). After PQ exposure, the content of hydroxyproline in lung tissue increased gradually and reached the peak value on the 28th day. Compared with the PQ group, the contents of hydroxyproline in the PQ+PFD 200 group decreased at the 7th, 14th and 28th day, and the contents of malondialdehyde decreased at the 3rd and 7th day, the differences were statistically significant (P<0.05). The levels of TNF-α, IL-6 in rat serum and lung tissue reached the peak value on the 7th day after PQ exposure, and the levels of TGF-β1, FGF-B and IGF-1 in rat serum and lung tissue reached the peak value on the 14th day after PQ exposure, and the level of PDGF-AB in rat serum and lung tissue reached the peak value on the 28th day after PQ exposure. Compared with PQ group, the level of serum IL-6 in PQ+PFD 200 group decreased significantly on the 7th day, and serum TGF-β1, FGF-B, PDGF-AB and IGF-1 on the 14th and 28th day were decreased significantly (P<0.05). The levels of TNF-α, IL-6 in lung tissue of rats in PQ+PFD 200 group on the 7th day decreased significantly, and the levels of TGF-β1, FGF-B and IGF-1 in lung tissue of rats on the 14th day were significantly decreased, and the level of PDGF-AB in lung tissue of rats on the 28th day were significantly decreased (P<0.05) . Conclusion: PFD partially alleviates the PQ-induced lung inflammation and fibrosis by inhibiting oxidative stress, reducing the levels of pro-inflammatory and pro-fibrotic cytokines in serum and lung tissue, but does not affect the concentrations of PQ in serum and lung tissue.

Dimethyl fumarate attenuates paraquat-induced pulmonary oxidative stress, inflammation and fibrosis in mice

Paraquat (PQ) is the most important cationic bipyridyl herbicide in the agricultural industry, which is very toxic to humans and animals and causes disruption in many organs, mainly in the lungs. Dimethyl fumarate (DMF) is an immune-modulating drug used in the treatment of multiple sclerosis and psoriasis shows antioxidant, anti-inflammatory, and antifibrotic effects. In this study, the ameliorative effects of DMF (10, 30 and 100 mg/kg, orally) on PQ (30 mg/kg) model of lung damage were evaluated in male mice. DMF was given daily for 7 days and PQ was administrated in the fourth day in a single dose. On the eighth day, the animals were sacrificed, and their lung tissue were removed. The results indicated that DMF can ameliorate PQ-induced the significant increase in lung index, hydroxyproline, as well as TBARS, TGF-β, NF-κB and decrease in the amount of total thiol, catalase, glutathione peroxidase, superoxide dismutase, Nrf-2, and INF-γ. The histopathological results confirmed indicated findings. The results showed that the protective effect of DMF on PQ-induced toxicity is mediated through antioxidant, anti-inflammatory and antifibrotic activities.

Mutual promotion of mitochondrial fission and oxidative stress contributes to mitochondrial-DNA-mediated inflammation and epithelial-mesenchymal transition in paraquat-induced pulmonary fibrosis.

Pulmonary fibrosis (PF) is one of the main causes of death in patients with paraquat (PQ) poisoning. This study aimed to evaluate the relationship between mitochondrial fission and oxidative stress in PQ-induced epithelial-mesenchymal transition (EMT) and PF. C57BL/6 mice and MLE-12 cells were exposed to PQ to construct a PF model in vivo and in vitro. Histological changes in the lungs were examined by hematoxylin and eosin (H&E) staining. Mitochondrial morphology was detected by MitoTracker® Deep Red FM or transmission electron microscopy (TEM). Western blotting and immunofluorescence were used to determine the expression of protein. The migration ability of the cells was detected by the cell scratch test. Mitochondrial DNA (mtDNA) levels were assessed by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect cytokine levels. Superoxide dismutase (SOD) activity and the levels of glutathione (GSH) and malondialdehyde (MDA) were detected by chemichromatometry. PQ exposure caused EMT and PF in vivo and in vitro. PQ destroyed mitochondrial structure and enhanced the expression of dynamin-related protein 1 (Drp1), which were accompanied by oxidative stress. Inhibiting mitochondrial fission using mitochondrial division inhibitor-1 (Mdivi-1), a selective inhibitor of Drp1, attenuated PQ-induced EMT and oxidative damage. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, reduced Drp1 expression, attenuated mitochondrial structure damage and inhibited PQ-induced EMT and PF. Both Mdivi-1 and NAC treatment markedly suppressed mtDNA release, the expression of Toll-like receptor 9 (TLR9) and phosphorylation (P)-NF-κB p65 as well as cytokines (interleukin 6 [IL-6], interleukin-1β [IL-1β], and tumor necrosis factor-α [TNF-α]) production. Mutual promotion of mitochondrial fission and oxidative stress contributes to EMT in PQ-induced PF, which is associated with the mtDNA/TLR9/NF-κB pathway.

PD-L1 upregulation promotes drug-induced pulmonary fibrosis by inhibiting vimentin degradation

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with high mortality and limited therapeutic options. The immune checkpoint PD1/PD-L1 axis is related to the pathogenesis of pulmonary fibrosis, and upregulated expression levels of PD-L1 have been demonstrated in IPF patients. However, the mechanism of PD-L1 in pulmonary fibrosis is not fully understood. Here, we demonstrated upregulated expression of PD-L1 in fibrotic lung tissues and sera of IPF patients. Bleomycin (BLM) treatment induced PD-L1 upregulation, EMT (Epithelial-Mesenchymal Transition) and fibrosis-like morphology changes in human pulmonary alveolar epithelial cells (HPAEpiCs). Silencing PD-L1 attenuated BLM-induced EMT and fibrosis-like morphology changes in HPAEpiCs. In addition, we identified that PD-L1 directly binds to vimentin and inhibits vimentin ubiquitination, thereby increasing vimentin levels in HPAEpiCs. Silencing of vimentin inhibited BLM- and PD-L1-induced fibrosis in HPAEpiCs. The correlation between PD-L1 and EMT or vimentin expression was further confirmed in clinical samples and animal models. Finally, we used BLM- and paraquat-induced pulmonary fibrosis animal models to confirm the anti-pulmonary fibrosis effects of PD-L1 silencing. Taken together, our findings suggest that upregulated PD-L1 stimulates EMT of alveolar epithelial cells by increasing vimentin levels by inhibiting vimentin ubiquitination, thereby contributing to pulmonary fibrosis.

E2F1 regulates miR-215-5p to aggravate paraquat-induced pulmonary fibrosis via repressing BMPR2 expression.

Pulmonary fibrosis is considered to be an irreversible lung injury, which can be caused by paraquat (PQ) poisoning. MiRNAs have been demonstrated crucial roles in pulmonary fibrosis caused by numerous approaches including PQ induction. The purpose of this study was to investigate the role and the underlying mechanism of miR-215 in PQ-induced pulmonary fibrosis. The cell and animal models of pulmonary fibrosis were established through PQ intervention. Cell viability was performed to test by MTT assay. Immunofluorescence assay was used to detect COL1A1 expression and its location. The relationships among E2F1, miR-215-5p, and BMPR2 were validated by dual luciferase reporter gene assay, chromatin immunoprecipitation and RNA-binding protein immunoprecipitation. Lung morphology was evaluated by hematoxylin and eosin staining. MiR-215-5p was upregulated in PQ-induced pulmonary fibrosis in vitro and in vivo. MiR-215-5p silencing relieved PQ-induced pulmonary fibrosis progression by enhancing cell viability and reducing the expression of fibrosis-related markers (COL1A1, COL3A1, and α-SMA). Mechanistically, miR-215-5p directly targeted BMRP2. BMPR2 knockdown abolished the suppressive effects of miR-215-5p knockdown on PQ-induced pulmonary fibrosis. In addition, E2F1 interacted with miR-215-5p promoter and positively regulated miR-215-5p expression. E2F1 downregulation reduced miR-215-5p level and promoted BMPR2 level via regulating TGF-β/Smad3 pathway, and then suppressed PQ-induced pulmonary fibrosis, whereas these effects were compromised by miR-215-5p sufficiency. MiR-215-5p was activated by E2F1 to repress BMPR2 expression and activate TGF-β/Smad3 pathway, which aggravated PQ-induced pulmonary fibrosis progression. Targeting the E2F1/miR-215-5p/BMPR2 axis might be a new approach to alleviate PQ-induced pulmonary fibrosis.

Pathophysiological Changes in Rhesus Monkeys with Paraquat-Induced Pulmonary Fibrosis.

PurposePulmonary fibrosis is a life-threatening lung disorder. A comprehensive understanding of the pathophysiological changes in the development of pulmonary fibrosis will lead to new insights into its treatment.MethodsWe used a paraquat (PQ)-induced rhesus monkey model of pulmonary fibrosis to comprehensively investigate the process of pulmonary fibrosis development. Rhesus monkeys were orally administered PQ at concentrations of 25 mg/kg, 40 mg/kg, and 80 mg/kg. The dose was given once. Behavior and clinical data, such as PQ concentration, arterial oxygen saturation, biochemical evaluation, lung histopathology, and medical imaging, were continuously observed.ResultsParaquat-exposed monkeys developed pulmonary fibrosis following an expected time course, especially at 25 mg/kg. CT images showed ground-glass lesions in the lung after 4 weeks, and pulmonary fibrosis persisted until the end of follow-up. Using pathological examination, the lung sustained collagen deposition and slight inflammatory cell infiltration. All rhesus monkeys had obvious inflammatory infiltration within 1 week according to the immunohistochemical results and the number of leukocytes in the blood. The CT results showed that pulmonary fibrosis had not formed, indicating that drugs with powerful anti-inflammatory ability are potential candidates for early pulmonary fibrosis treatment.ConclusionOur study describes the dynamic process of paraquat-induced pulmonary fibrosis in rhesus monkeys and provided a pathophysiological basis for the treatment of pulmonary fibrosis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00408-022-00572-9.

Time-resolved proteome and transcriptome of paraquat-induced pulmonary fibrosis

BackgroundsPulmonary fibrosis (PF) is a pathological state presenting at the progressive stage of heterogeneous interstitial lung disease (ILD). The current understanding of the molecular mechanisms involved is incomplete. This clinical toxicology study focused on the pulmonary fibrosis induced by paraquat (PQ), a widely-used herbicide. Using proteo-transcriptome analysis, we identified differentially expressed proteins (DEPs) derived from the initial development of fibrosis to the dissolved stage and provided further functional analysis. MethodsWe established a mouse model of progressive lung fibrosis via intratracheal instillation of paraquat. To acquire a comprehensive and unbiased understanding of the onset of pulmonary fibrosis, we performed time-series proteomics profiling (iTRAQ) and RNA sequencing (RNA-Seq) on lung samples from paraquat-treated mice and saline control. The biological functions and pathways involved were evaluated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis. Correlation tests were conducted on comparable groups 7 days and 28 days post-exposure. Differentially expressed proteins and genes following the same trend on the protein and mRNA levels were selected for validation. The functions of the selected molecules were identified in vitro. The protein level was overexpressed by transfecting gene-containing plasmid or suppressed by transfecting specific siRNA in A549 cells. The levels of endothlial-mesenchymal transition (EMT) markers, including E-cadherin, vimentin, FN1, and α-SMA, were determined via western blot to evaluate the fibrotic process. ResultsWe quantified 1358 DEPs on day 7 and 426 DEPs on day 28 post exposure (Fold change >1.2; Q value < 0.05). The top 5 pathways – drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, complement and coagulation cascades, chemical carcinogenesis, protein digestion and absorption — were involved on both day 7 and day 28. Several pathways, including tight junction, focal adhesion, platelet activation, and ECM-receptor interaction, were more enriched on day 28 than on day 7. Integrative analysis of the proteome and transcriptome revealed a moderate correlation of quantitative protein abundance ratios with RNA abundance ratios (Spearman R = 0.3950 and 0.2477 on days 7 and 28, respectively), indicating that post-transcriptional regulation plays an important role in lung injury and repair. Western blot identified that the protein expressions of FN1, S100A4, and RBM3 were significantly upregulated while that of CYP1A1, FMO3, and PGDH were significantly downregulated on day 7. All proteins generally recovered to baseline on day 28. qPCR showed the mRNA levels of Fn1, S100a4, Rbm3, Cyp1a1, Fmo3, and Hpgd changed following the same trend as the levels of their respective proteins. Further, in vitro experiments showed that RBM3 was upregulated while PGDH was downregulated in an EMT model established in human lung epithelial A549 cells. RBM3 overexpression and PGDH knockout could both induce EMT in A549 cells. RBM3 knockout or PGDH overexpression had no reverse effect on EMT in A549 cells. ConclusionsOur proteo-transcriptomic study determined the proteins responsible for fibrogenesis and uncovers their dynamic regulation from lung injury to repair, providing new insights for the development of biomarkers for diagnosis and treatment of fibrotic diseases.

Barbaloin attenuates pulmonary fibrosis through TGF-β1/Smads/p38 pathway.

Barbaloin is one of the main bioactive ingredients extracted from Aloe vera, which has the property of protecting the lung from LPS-induced acute injury; however, the anti-pulmonary fibrosis effect of barbaloin is still unknown. Herein, we present novel data showing the anti-pulmonary fibrosis effect of barbaloin and revealing the possible molecular mechanism. In vivo experiment, oral administration of barbaloin was investigated through paraquat-induced pulmonary fibrosis in mice. In vitro experiment, epithelial-mesenchymal transition (EMT) process and TGF-β1 pathway were investigated in A549 cells for exploring the anti-fibrosis molecular mechanism of barbaloin. Results showed that barbaloin could improve pulmonary fibrosis through improving physiological routine indexes and histopathological lesions of mice in a dose-dependent manner. Hydroxyproline, collagen I, N-cadherin and α-SMA levels were significantly suppressed. Besides, pro-inflammatory cytokines were also improved. In vitro experiment, barbaloin could inhibit the process of EMT through repressing α-SMA, collagen I and N-cadherin and increasing E-cadherin. In addition, barbaloin could repress the expression of p-Smad2/3 and then suppress the process of EMT through intervening TGF-β1-induced canonical pathway. Moreover, MMP-2 and MMP-9 were also inhibited by barbaloin via repressing phosphorylation of p38 through TGF-β1-induced non-canonical axis. Our findings reveal the anti-pulmonary fibrosis effect of barbaloin in vivo and in vitro for the first time. These results indicate that barbaloin may be a promising clinical candidate drug against pulmonary fibrosis.

Wnt-induced secreted proteins-1 play an important role in paraquat-induced pulmonary fibrosis

BackgroundThe objective of this article is to observe the expression of Wnt-induced secreted proteins-1 (WISP1) in paraquat (PQ)-induced pulmonary fibrosis (PF) to explore the role of WISP1.MethodsHealthy individuals were included in the control group. Patients who had acute lung injury or PF were included in the PF group. Venous blood samples were collected from the patients on days 1 and 3 following PQ poisoning to detect the expression levels of the WISP1 gene and protein concentration. Any changes in the patients’ blood gas analysis index were reviewed. In addition, chest computed tomography (CT) and x-ray images were observed to evaluate the relationship between WISP1 expression and disease severity.ResultsThe expression of the WISP1 gene and the serum WISP1 protein concentration were higher in patients with PQ poisoning combined with PF than in patients without PF (P < 0.01). Serum PQ concentration was positively correlated with WISP1 gene expression (r = 0.621, P < 0.01), and serum WISP1 protein concentration (r = 0.596, P < 0.01) was considered a risk factor [odds ratio (OR) = 4.356, P < 0.05] for PQ-induced PF. Concurrently, the results of the adjusted and non-adjusted OR value for WISP1 gene expression and WISP1 protein concentration on day 1 was, respectively, as follows: OR = 12.797, 95% confidence interval (CI) (2.478–66.076), P = 0.002, OR’ = 11.353, P = 0.005; and OR = 1.545, 95% CI (1.197–1.995), P = 0.001, OR’ = 1.487, P = 0.003. The CT scan of a 20-year-old male with PQ-induced PF (20 ml) was observed, and it showed a typical hyaline-like lesion in the lungs on day 22 after poisoning; on day 33 after poisoning, the lungs showed localised consolidation combined with air bronchography.ConclusionThe expression of WISP1 was higher in the patients with PQ-induced PF compared with the patients without PF. Accordingly, WISP1 plays an important role in PQ-induced PF.

Time-Resolved Proteome and Transcriptome of Paraquat-Induced Pulmonary Fibrosis

Time-Resolved Proteome and Transcriptome of Paraquat-Induced Pulmonary Fibrosis

Synthesis and anti-inflammatory activities of glycyrrhetinic acid derivatives containing disulfide bond

A series of glycyrrhetinic acid (GA, aglycone of glycyrrhizic acid) derivatives containing disulfide bond were synthesized and their anti-inflammatory and anti-fibrosis activities were evaluated in vivo and in vitro. Among them, compound 7 displayed the highest toxicity to all the tested cell lines including macrophages. Compounds 3 and 4 showed higher activities than GA in the cell and animal model. In the anti-inflammatory tests, compounds 3 and 4 down-regulated the expressions of several inflammatory factors, such as HMGB1, TLR4, IL-1β, TNF-α and TGF-β1 in LPS-treated RAW264.7 cells in a dose-dependent manner. Compounds 3 and 4 at 30 µM respectively reduced the levels of HMGB1 in the LPS group to 42.7% and 38.2%. In addition, the level of TLR4 decreased to close to that of control group when treated by compound 4 at the concentration of 30 µM. In the process of anti-fibrosis tests using TGF-β1-induced A549 cell line as the model, compounds 3 and 4 also decreased the expression levels of Col1 and α-SMA in a dose-dependent manner. Compound 3 and 4 at 30 µM respectively reduced the expression of α-SMA level by 2.2-fold and 2.6-fold compared to the TGF-β1-treated control group. Moreover, they influenced the ROS level and mitochondrial membrane potential (MMP) in A549 cells. In the paraquat-induced pulmonary fibrosis mice model, the symptoms of inflammation and fibrosis of mice were alleviated after administration of compound 3 or 4. The above results suggest that compounds 3 and 4 may be promising candidates for inflammation and lung fibrosis treatment.