Lung Fibrosis In Rats Research Articles

Pulmonary damage induction upon Acrylic amide exposure via activating miRNA-223–3p and miRNA-325–3p inflammasome/pyroptosis and fibrosis signaling pathway: New mechanistic approaches of A green-synthesized extract

Exposure to acrylic amide (AD) has garnered worldwide attention due to its potential adverse health effects, prompting calls from the World Health Organization for intensified research into associated risks. Despite this, the relationship between oral acrylic amide (acrylamide) (AD) exposure and pulmonary dysfunction remains poorly understood. Our study aimed to investigate the correlation between internal oral exposure to AD and the decline in lung function, while exploring potential mediating factors such as tissue inflammation, oxidative stress, pyroptosis, and apoptosis. Additionally, we aimed to evaluate the potential protective effect of zinc oxide nanoparticles green-synthesized moringa extract (ZNO-MONPs) (10 mg/kg b.wt) against ACR toxicity and conducted comprehensive miRNA expression profiling to uncover novel targets and mechanisms of AD toxicity (miRNA 223–3 P and miRNA 325–3 P). Furthermore, we employed computational techniques to predict the interactions between acrylic amide and/or MO-extract components and tissue proteins. Using a rat model, we exposed animals to oral acrylamide (20 mg/kg b.wt for 2 months). Our findings revealed that AD significantly downregulated the expression of miRNA 223–3 P and miRNA 325–3 P, targeting NLRP-3 & GSDMD, respectively, indicating the induction of pyroptosis in pulmonary tissue via an inflammasome activating pathway. Moreover, AD exposure resulted in lipid peroxidative damage and reduced levels of GPX, CAT, GSH, and GSSG. Notably, AD exposure upregulated apoptotic, pyroptotic, and inflammatory genes, accompanied by histopathological damage in lung tissue. Immunohistochemical and immunofluorescence techniques detected elevated levels of indicative harmful proteins including vimentin and 4HNE. Conversely, concurrent administration of ZNO-MONPs with AD significantly elevated the expression of miRNA 223–3 P and miRNA 325–3 P, protecting against oxidative stress, apoptosis, pyroptosis, inflammation, and fibrosis in rat lungs. In conclusion, our study highlights the efficacy of ZNO-MONPs NPs in protecting pulmonary tissue against the detrimental impacts of foodborne toxin AD.

Shenqi Chongcao Formula ameliorates inflammatory response in rats with pulmonary fibrosis by activating the ASS1/src/STAT3 signaling pathway

To observe the effect of Shenqi Chongcao (SQCC) Formula on the ASS1/src/STAT3 signaling pathway in a rat model of lung fibrosis and explore its therapeutic mechanism. A total of 120 male SD rats were divided equally into 5 groups, including a blank control group with saline treatment and 4 groups of rat models of idiopathic pulmonary fibrosis induced by intratracheal instillation of bleomycin. One day after modeling, the rat models were treated with daily gavage of 10 mL/kg saline, SQCC decoction (0.423 g/kg), pirfenidone (10 mL/kg), or intraperitoneal injection of arginine deiminase (ADI; 2.25 mg/kg, every 3 days) for 28 days. After the treatments, the lung tissues of the rats were collected for calculating the lung/body weight ratio, observing histopathology using HE and Masson staining, and analyzing the inflammatory cells in BALF using Giemsa staining. Serum chemokine ligand 2 (CCL2) and transforming growth factor-β1 (TGF-β1) levels were measured with ELISA. The protein expressions of src, p-srcTry529, STAT3, and p-STAT3Try705 and the mRNA expressions of ASS1, src and STAT3 in the lung tissues were detected using Western blotting and RT-qPCR. The neutrophil, macrophage and lymphocyte counts and serum levels of CCL2 and TGF-β1 were significantly lower in SQCC, pirfenidone and ADI treatment groups than in the model group at each time point of measurement (P < 0.05). P-srcTry529 and p-STAT3Try705 protein expression levels and ASS1, src, and STAT3 mRNA in the lung tissues were also significantly lower in the 3 treatment groups than in the model group (P < 0.05). SQCC Formula can alleviate lung fibrosis in rats possibly by activating the ASS1/src/STAT3 signaling pathway in the lung tissues.

Combination of losartan with pirfenidone: a protective anti-fibrotic against pulmonary fibrosis induced by bleomycin in rats

Pirfenidone (PFD), one acceptable medication for treating idiopathic pulmonary fibrosis (IPF), is not well tolerated by patients at full doses. Hence, employing of some approaches such as combination therapy may be applicable for increasing therapeutic efficacy of PFD. Losartan (LOS), an angiotensin II receptor antagonist, could be a suitable candidate for combination therapy because of its stabilizing effect on the pulmonary function of IPF patients. Therefore, this study aimed to investigate the effects of LOS in combination with PFD on bleomycin (BLM)-induced lung fibrosis in rats. BLM-exposed rats were treated with LOS alone or in combination with PFD. The edema, pathological changes, level of transforming growth factor-β (TGF-β1), collagen content, and oxidative stress parameters were assessed in the lung tissues. Following BLM exposure, the inflammatory response, collagen levels, and antioxidant markers in rat lung tissues were significantly improved by PFD, and these effects were improved by combination with LOS. The findings of this in vivo study suggest that the combined administration of PFD and LOS may provide more potent protection against IPF than single therapy through boosting its anti-inflammatory, anti-fibrotic, and anti-oxidant effects. These results hold promise in developing a more effective therapeutic strategy for treating of lung fibrosis.

Hydroxytyrosol Alleviates Methotrexate-Induced Pulmonary Fibrosis in Rats: Involvement of TGF-β1, Tissue Factor, and VEGF.

Methotrexate (MTX) is an indispensable drug used for the treatment of many autoimmune and cancerous diseases. However, its clinical use is associated with serious side effects, such as lung fibrosis. The main objective of this study is to test the hypothesis that hydroxytyrosol (HT) can mitigate MTX-induced lung fibrosis in rats while synergizing MTX anticancer effects. Pulmonary fibrosis was induced in the rats using MTX (14 mg/kg/week, per os (p.o.)). The rats were treated with or without HT (10, 20, and 40 mg/kg/d p.o.) or dexamethasone (DEX; 0.5 mg/kg/d, intraperitoneally (i.p.)) for two weeks concomitantly with MTX. Transforming growth factor beta 1 (TGF-β1), interleukin-4 (IL-4), thromboxane A2 (TXA2), vascular endothelial growth factor (VEGF), 8-hydroxy-2-deoxy-guanosine (8-OHdG), tissue factor (TF) and fibrin were assessed using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and RT-PCR. Pulmonary fibrosis was manifested by an excessive extracellular matrix (ECM) deposition and a marked increase in TGF-β1 and IL-4 in lung tissues. Furthermore, cotreatment with HT or dexamethasone (DEX) significantly attenuated MTX-induced ECM deposition, TGF-β1, and IL-4 expression. Similarly, HT or DEX notably reduced hydroxyproline contents, TXA2, fibrin, and TF expression in lung tissues. Moreover, using HT or DEX downregulated the gene expression of TF. A significant decrease in lung contents of VEGF, IL-8, and 8-OHdG was also observed in HT + MTX- or DEX + MTX -treated animals in a dose-dependent manner. Collectively, the results of our study suggest that HT might represent a potential protective agent against MTX-induced pulmonary fibrosis.

Modified Qing-Zao-Jiu-Fei decoction attenuated pulmonary fibrosis induced by bleomycin in rats via modulating Nrf2/NF-κB and MAPKs pathways

BackgroundQing-Zao-Jiu-Fei Decoction (QZJFD) is a famous herbal formula commonly prescribed for the treatment of lung-related diseases in the ancient and modern times. Trichosanthis Fructus (TF) and Fritillariae Thunbergii Bulbus (FTB) are widely used for treatment of cough and pulmonary disease. In order to identify a more effective formula for treatment of pulmonary fibrosis, we intend to add TF and FTB in QZJFD to form a modified QZJFD (MQZJFD). In this study, we aims to explore MQZJFD as an innovative therapeutic agent for pulmonary fibrosis using bleomycin (BLM)-treated rats and to unravel the underlying molecular mechanisms.MethodsBLM was given to SD rats by intra-tracheal administration of a single dose of BLM (5 mg/kg). QZJFD (3 g/kg) and MQZJFD (1, 2 and 4 g/kg) was given intragastrically daily to rats for 14 days (from day 15 to 28) after BLM administration for 14 consecutive days.ResultsMQZJFD was found to contain 0.29% of amygdalin, 0.020% of lutin, 0.077% of glycyrrhizic acid and 0.047% of chlorogenic acid. BLM treatment could induce collagen deposition in the lung tissues of rats, indicating that the pulmonary fibrosis rat model had been successfully established. MQZJFD have better effects than the original QZJFD in reducing the pulmonary structure damage and collagen deposition of rat lung fibrosis induced by BLM. MQZJFD could reduce the hydroxyproline content in lung tissues of BLM-treated rats. The biomarkers of fibrosis such as matrix metalloproteinase 9 (MMP9), collagen I and α-smooth muscle actin (α-SMA) were remarkably reduced after treatment with MQZJFD. MQZJFD also have anti-oxidant stress effects by inhibiting the level of malondialdehyde (MDA), but enhancing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the level of glutathione (GSH) in the lung tissues of BLM-treated rats. Moreover, the MQZJFD markedly suppressed the over expressions of p-p65/p65 and p-IκBα/IκBα, but upregulated the Nrf2. MQZJFD also suppressed the protein expressions of p-ERK1/2/ERK1/2, p-p38/p38 and p-JNK/JNK in the lung tissues of BLM-treated rats.ConclusionsMQZJFD could improve the pulmonary fibrosis induced by BLM in rats via inhibiting the fibrosis and oxidative stress via suppressing the activation of NF-κB/Nrf2 and MAPKs pathways.

Cinnamic acid mitigates methotrexate-induced lung fibrosis in rats: comparative study with pirfenidone

PurposeLung fibrosis is a heterogeneous lung condition characterized by excessive accumulation of scarred tissue, leading to lung architecture destruction and restricted ventilation. The current work was conducted to examine the probable shielding influence of cinnamic acid against lung fibrosis induced by methotrexate.MethodsRats were pre-treated with oral administration of cinnamic acid (50 mg/kg/day) for 14 days, whereas methotrexate (14 mg/kg) was orally given on the 5th and 12th days of the experiment. Pirfenidone (50 mg/kg/day) was used as a standard drug. At the end of the experiment, oxidative parameters (malondialdehyde, myeloperoxidase, nitric oxide, and total glutathione) and inflammatory mediators (tumor necrosis factor-α and interleukin-8), as well as transforming growth factor-β and collagen content, as fibrosis indicators, were measured in lung tissue.ResultsOur results revealed that cinnamic acid, as pirfenidone, effectively prevented the methotrexate-induced overt histopathological damage. This was associated with parallel improvements in oxidative, inflammatory, and fibrotic parameters measured. The outcomes of cinnamic acid administration were more or less the same as those of pirfenidone. In conclusion, pre-treatment with cinnamic acid protects against methotrexate-induced fibrosis, making it a promising prophylactic adjuvant therapy to methotrexate and protecting against its possible induction of lung fibrosis.

Betulin-rich hydroalcoholic extract of Daphne oleoides attenuates bleomycin-induced pulmonary fibrosis in rat

Background and objectivePulmonary fibrosis (PF) is a chronic and progressive respiratory disease representing the final stage of lung inflammatory disorders. Reactive oxygen species (ROS), an essential factor in the formation and progression of pulmonary fibrosis, are a significant adverse effect of Bleomycin (BLM). Antioxidant activities have been found in Daphne oleoides. In this study, we attempted to explore the function of hydroalcoholic extract of Daphne oleoides (D. oleoides) and Betulin in inhibiting bleomycin (BLM)-induced pulmonary fibrosis in rat". Materials and methodsThe current experimental study used 36 male Wistar rats (180–220). Following a random process, the animals were divided into six groups six (n = 6). Group, I (the control group) received normal saline, while Group II (the hazardous group) received intratracheal BLM (7.5 units per kg). Following the administration of BLM, Groups V and VI received daily doses of vitamin E (500 mg/kg/d, p.o.) and Betulin (10 mg kg/d, p.o.), whereas Groups III and IV received daily doses of Daphne oleoides extract (300 and 600 mg/kg/d, p.o.). Then, blood samples from the hearts of the animals were taken to assess the plasma concentrations of nitric oxide (NO) and malondialdehyde (MDA). Finally, the rats were euthanized, and the lung tissues were taken out for histological analysis and assessments of the levels of lung hydroxyproline (HP), ferric-reducing ability (FRAP), NO, Glutathione Concentration (GSH), thiol content (tSH) and MDA. FindingsElevated lung index, lung hydroxyproline, NO, and MDA plasma levels, and a reduction in total body thiol content (tSH) in the group receiving BLM were evidence of pulmonary toxicity. Treatment with D. oleoides extracts, Betulin, and Vit E, especially at 600 mg/kg, led to a marked reduction in the above parameters compared with the BLM-received group (p < 0.01). Histological Analysis of the BLM-treated group showed a considerable Lung injury with interstitial infiltration, collapsed alveolar spaces, and alveolar septal thickening. These changes were mitigated with D. oleoides 600, Betulin-, and vitamin E. These changes were mitigated with D. oleoides 600, Betulin-, and vitamin E. ConclusionThese findings suggest that D. oleoides and Betulin prevent bleomycin-induced lung fibrosis in rats by decreasing inflammatory and antioxidant markers. Daphne oleoides, therefore, have the potential to be used therapeutically to treat pulmonary fibrosis.

Repurposing of carvedilol to alleviate bleomycin-induced lung fibrosis in rats: Repressing of TGF-β1/α-SMA/Smad2/3 and STAT3 gene expressions

Idiopathic pulmonary fibrosis (IPF) is the most widely studied interstitial lung disease. IPF eventually leads to respiratory insufficiency, lung cancer, and death. Carvedilol (CAR) is a third-generation β-adrenergic receptor antagonist with an α1-blocking effect. CAR demonstrates antifibrotic activities in various experimental models of organ fibrosis. Aims: This work is designed to explore the possible alleviating effects of CAR on bleomycin (BLM)-induced lung fibrosis in rats. Main methods: The BLM rat model of lung fibrosis was achieved by intratracheal delivery of a single dose of 5 mg/kg of BLM. Seven days following BLM injection, either prednisolone or CAR was orally administered at doses of 10 mg/kg once daily for 21 days to the rats. The actions of CAR were evaluated by lung oxidant/antioxidant parameters, protein concentration and total leucocyte count (TLC) in bronchoalveolar lavage fluid (BALF), fibrosis regulator-related genes along with the coexistent lung histological changes. Key findings: CAR effectively decreased lung malondialdehyde level, increased superoxide dismutase activity, declined both protein concentration and TLC in BALF, downregulated TGF-β1/α-SMA/Smad2/3 and STAT3 gene expressions, and repaired the damaged lung tissues. Significance: CAR conferred therapeutic potential against BLM-induced lung fibrosis in rats, at least in part, to its antioxidant, anti-inflammatory, and antifibrotic activities. CAR could be utilized as a prospective therapeutic option in patients with lung fibrosis in clinical practice.

Protective Effect of Tunisian Red Seaweed (Corallina officinalis) Against Bleomycin-Induced Pulmonary Fibrosis and Oxidative Stress in Rats.

Idiopathic pulmonary fibrosis is a chronic and progressive respiratory disease whose diagnosis and physiopathogenesis are still poorly understood and for which, until recently, there were no effective treatments. Over the past few decades, many studies have demonstrated that marine macroalgae such as red seaweeds are potential alternative sources of useful bioactive compounds possessing various physiological and biological activities. The present study was aimed to investigate the effect of Corallina officinalis aqueous extract (COAE) against bleomycin (BLM)-induced lung fibrosis in rat. Thus, Wistar rats were divided into 4 groups of 10 each: control, BLM (2mg/kg), BLM/COAE-150mg/kg and BLM/COAE-300mg/kg once a day for 21 days. Obtained results showed that COAE is rich in phenolic compounds and exhibited relatively high antioxidant activity. COAE might significantly reduce the damage caused by BLM by rewarding the decline in weight and pulmonary index in rats given only BLM. Moreover, lungs, liver and kidneys lipid peroxidation, and sulfhydryl group levels were reversed significantly in a dose-dependent manner in the COAE-treated groups. BLM decreased superoxide dismutase (SOD) and catalase (CAT) activities, while COAE administration increased the antioxidant enzyme activities. Histopathologically, COAE attenuates the severity of the inflammatory lungs state caused by instillation of BLM in rats. These findings suggest that COAE can be a potential therapeutic candidate against BLM-induced lung fibrosis.

SiO2 dust induces inflammation and pulmonary fibrosis in rat lungs through activation of ASMase/Ceramide pathway.

The role of ASMase/Ceramide signaling pathway in the development of silicosis needs to be verified by in vivo experiments. We investigated the role of the ASMase/Ceramide signaling pathway in the progression of silicosis and the effect of desipramine (DMI)(1mg/mL) on the development of silicosis, by establishing a silica(1mL, 50mg/mL) dust-contaminated rat silicosis model and administering the ASMase inhibitor, DMI, to the dust-contaminated rats. The results showed that levels of IL-1β and IL-6 were increased in the lung tissues of the rats in the dust-contaminated group at the initial stage after dusting, the inflammatory cell aggregation in the lung tissue was increased. With time progression, the hydroxyproline content in the lung tissue increased, and α-SMA, collagen I and vimentin substantially increased, suggesting that silicosis were formed in the lung tissue of the rats 28 days after SiO2 dust treatment. Moreover, the levels of ASMase, ceramide and S1P were increased in the lung tissue of rats. The expression of β-catenin, fibronectin and caspase-3 protein was increased, and E-cadherin protein expression was decreased in the lung tissue of the rats in the late stage of dust contamination. The ASMase and ceramide in the lung tissues of the rats in the DMI intervention group were reduced, as were the lung tissue inflammation levels, collagen expression, and lung fibrosis. These results suggest that SiO2 dust may activate the ASMase/ceramide signaling pathway in rat lung tissue, promoting pulmonary fibrosis. DMI inhibited these activation, attenuated apoptosis and blocked epithelial-mesenchymal transition and halted silica dust-induced silicofibrosis.

TLR4/ MyD88/NF-κB signaling pathway involved in the protective effect of diacerein against lung fibrosis in rats.

Pulmonary fibrosis (PF) is an inescapable problem. Diacerein, a chondro-protective drug, has antioxidant and anti-inflammatory effects. Its effect on PF injury has not yet been fully clarified. Therefore, the current study aimed to detect its protective effect on lung tissue with the explanation of possible underlying mechanisms. Adult male albino rats were assigned to four groups: control group, diacerein control group, PF non-treated group, and PF diacerein pretreated group. Lung tissue oxidative stress parameters, inflammatory biomarkers mainly Toll-like receptors-4 (TLR4), and myeloid differentiation factor 88 (MyD88) levels were determined. Histopathological examination of lung tissue and immunohistochemical studies of nuclear factor-kappa B (NF-κB), and transforming growth factor- β (TGF-β) were also done. Diacerein pretreatment has the ability to restore the PF damaging effect, proved by the reduction of the oxidative stress and lung tissue inflammation via downregulation of TLR4/NF-κB signaling pathway together with the restoration of TGF-β level and improvement of the histopathological and immunohistochemical study findings in the lung tissue. These results suggested the protective effect of diacerein on PF relies on its antioxidant and anti-inflammatory effects reducing TLR4/NF-κB signaling pathway.

The targeted delivery of chitosan nanoparticles to treat indoxacarb: induced lung fibrosis in rats

BackgroundThis study evaluated the effects of chitosan nanoparticles (Ch-NPs) on indoxacarb (INDOX)-induced pulmonary fibrosis in in vivo and in vitro models. In in vivo studies, 40 male albino rats were randomly divided into four groups (10 rats/group): Group 1, normal control; Group 2, INDOX (600 mg/kg b.w.); Group 3, Ch-NPs (2 mg/kg b.w.); and Group 4, Ch-NPs + INDOX. Characterization of Ch-NPs was done measuring dynamic light scattering, zeta potential, Fourier-transform infrared spectroscopy, transmission electron microscopy, and antioxidant activity studies after various Ch-NPs treatments. From in vitro studies, the impact of Ch-NPs on A549 lung carcinoma cell proliferation was also examined.ResultsOur data indicated that INDOX provoked considerable lung damage as indicated by decreased antioxidant enzyme levels of superoxide dismutase and glutathione peroxidase, increased production of nitric oxide and malondialdehyde serum levels, elevated myeloperoxidase activity, increased hydroxyproline and cytokeratin-19 serum levels, and significantly upregulated matrix metallopeptidase-9 and microRNA-101 gene expression levels when compared with controls. Furthermore, histopathological and immunohistochemical investigations of cyclooxygenase-2 in the lung tissue revealed marked inflammation, severe fibrosis, and neutrophil infiltration. Critically, Ch-NPs treatment significantly reversed INDOX-induced changes in lung biochemical, histopathological, and immunohistochemical outcomes.ConclusionTherefore, Ch-NPs may function as potential therapeutic drugs for lung fibrosis owing to their antioxidant, anti-inflammatory, and antifibrotic activities with neutrophil infiltration.

Exposure Time of Silica Dust and the Incidence of Oxidative Stress, Inflammation, and Fibrosis in Rat Lungs

BACKGROUND: Until now, exposure to silica dust is still a health problem worldwide. Silica exposure in the lungs will cause pulmonary fibrosis which is initiated by inflammation. However, the results of several studies regarding the duration of inflammation and fibrosis are still inconsistent. There was a role of oxidative stress in silicosis, but there were also inconsistencies in terms of when oxidative stress occurs in silica exposure. AIM: This study aimed to study the toxic effects of silica dust exposure by looking at the picture of inflammation and fibrosis and malondialdehyde (MDA) levels in lung tissue during the observation period of 7 days, 14 days, 21 days, and 28 days. METHODS: This study used a randomized post-test only control group design. The research sample was male Wistar rat (Rattus norvegicus), aged 6–10 weeks, body weight 150–200 g (divided into 5 groups: Control group, day 7 group, group day 14, group day 21, and group day 28). We administered silica suspension through intratracheal injection of 30 mg/rat on 0.5 mL of volume. Examination of MDA level was using the ELISA technique; histopathological examination of the liver used hematoxylin-eosin (HE) staining to determine inflammation and fibrosis. Statistical test using one-way ANOVA or Kruskal–Wallis followed by post hoc test. RESULTS: The results of our study found that intratracheal silica exposure increased MDA levels on the 7th day, increased the accumulation of collagen from the 14th day, and increased the pulmonary inflammation score on the 14th day (p &lt; 0.05). CONCLUSIONS: It was concluded that silica exposure caused significant oxidative stress on day 7 as well as significant inflammation and pulmonary fibrosis on day 14.

Bioavailability Enhancement of Cepharanthine via Pulmonary Administration in Rats and Its Therapeutic Potential for Pulmonary Fibrosis Associated with COVID-19 Infection.

Cepharanthine (CEP) has excellent anti-SARS-CoV-2 properties, indicating its favorable potential for COVID-19 treatment. However, its application is challenged by its poor dissolubility and oral bioavailability. The present study aimed to improve the bioavailability of CEP by optimizing its solubility and through a pulmonary delivery method, which improved its bioavailability by five times when compared to that through the oral delivery method (68.07% vs. 13.15%). An ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method for quantification of CEP in rat plasma was developed and validated to support the bioavailability and pharmacokinetic studies. In addition, pulmonary fibrosis was recognized as a sequela of COVID-19 infection, warranting further evaluation of the therapeutic potential of CEP on a rat lung fibrosis model. The antifibrotic effect was assessed by analysis of lung index and histopathological examination, detection of transforming growth factor (TGF)-β1, interleukin-6 (IL-6), α-smooth muscle actin (α-SMA), and hydroxyproline level in serum or lung tissues. Our data demonstrated that CEP could significantly alleviate bleomycin (BLM)-induced collagen accumulation and inflammation, thereby exerting protective effects against pulmonary fibrosis. Our results provide evidence supporting the hypothesis that pulmonary delivery CEP may be a promising therapy for pulmonary fibrosis associated with COVID-19 infection.

Coomassie brilliant blue G-250 dye attenuates bleomycin-induced lung fibrosis by regulating the NF-κB and NLRP3 crosstalk: A novel approach for filling an unmet medical need

Pulmonary fibrosis (PF) is a life-threatening disorder with a very poor prognosis. Because of the complexity of PF pathological mechanisms, filling such an unmet medical need is challenging. A number of pulmonary diseases have been linked to the activation of NF-κB and the NLRP3 inflammasome. Coomassie brilliant blue G-250 (CBBG) is proved to be a safe highly selective P2×7R antagonist with promising consequent inactivation of NLRP3 inflammasome. This is the first report to investigate the effect of CBBG on the bleomycin-induced lung fibrosis in rats. Our findings revealed that CBBG resulted in a significant improvement in histological features and oxidative status biomarkers of bleomycin-exposed lung tissue. Additionally, CBBG repressed collagen deposition as indicated after the analysis of hydroxyproline, TGF-β, PDGF-BB, TIMP-1, MMP-9, Col1a1, SMA and ICAM-1. It also exhibited anti-inflammatory potential as revealed by the determination of TNF-α, IL-1β, IL-18, MCP-1 in the lung tissue. In the bronchoalveolar lavage, the total protein and the LDH activity were substantially reduced. The lung protective effects of CBBG might be attributed on the one hand to the inhibition of NLRP3 inflammasome and on the other hand to the inactivation of NF-κB. Decreased levels of phospho-p65 and its DNA-binding activity as well as the analysis of TLR4 confirmed NF-κB inactivation. Caspase-1 activity is suppressed as a consequence of inhibiting NLRP3 inflammasome assembly. To conclude, CBBG may act as a primary or adjuvant therapy for the management of PF and therefore it may pose an opportunity for a novel approach to an unmet medical need.

Protective effect of dapsone against bleomycin-induced lung fibrosis in rat

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial disease of the lung tissue that causes symptoms such as coughing and asthma. It is caused by inflammatory factors and oxidative stress. In vivo model of IPF is induced by bleomycin (BLM,) a chemotherapeutic agent. We have investigated the effect of dapsone on bleomycin-induced IPF in adult male Wistar rats due to its anti-inflammatory and anti-oxidative stress effects. The animals were randomly divided into 5 groups (Control, BLM, BLM + dapsone 1, BLM + Dapsone 3, BLM + Dapsone 10). The control group received normal water and food. In the fibrosis group, bleomycin (BLM) (5 mg/kg) was used to induce pulmonary fibrosis by intratracheal administration. Three groups of animals were treated daily with single doses of 1, 3, and 10 mg dapsone by intraperitoneal injection 1 h after receiving BLM for 2 weeks. The activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and oxidative stress markers such as myeloperoxidase (MPO), malondialdehyde (MDA), protein carbonyl (PC) and nitrite were measured to evaluate bleomycin and therapeutic effect of dapsone. The histological assays of lung tissues were done by Hematoxylin-eosin (H & E) and Masson's trichrome staining. BLM reduced the activity of oxidative enzymes and increased the oxidative stress markers, while treatment with dapsone has reversed the results. In addition, the total number of cells as inflammatory cells such as neutrophils and eosinophils were examined. It has been indicated BLM increased these cells, and dapsone decreased them. The results of H & E and Masson's trichrome staining showed that dapsone reduced inflammation and alveolar wall thickness and BLM-induced pulmonary fibrosis. According to the findings of this study, dapsone seems to have therapeutic effects on pulmonary fibrosis through its anti-inflammatory and anti-oxidative stress properties and reduction of the toxic effects of bleomycin.

Connective-Tissue Growth Factor Contributes to TGF-β1-induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and in the lung. Connective-tissue growth factor (CTGF) exacerbates pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we demonstrate upregulation of CTGF in a rat lung fibrosis model induced by an adenovirus vector encoding active TGF-β1 (AdTGF-β1). We show that CTGF is also upregulated in patients with idiopathic pulmonary fibrosis. Expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on Days 7 and 14 as well as endothelial cells sorted from fibrotic lungs on Days 14 and 28. These findings suggest contributions of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases profibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, the fibrotic extracellular matrix upregulated CTGF expression, compared with the normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for treatment of pulmonary fibrosis.

1β-Hydroxyalantolactone from Inulae Flos alleviated the progression of pulmonary fibrosis via inhibiting JNK/FOXO1/NF-κB pathway

Inulae Flos was widely distributed throughout Europe, Africa, and Asia, and was commonly used as a folk medicine in clinic for treating various respiratory diseases, including cough, asthma, bronchitis, pulmonary fibrosis, and pneumonia. However, the ingredients responsible for the pharmacology effects of I. Flos and the underlying mechanisms remain unclear. In this study, the effects of 16 known sesquiterpene lactones and flavonoids from I. Flos on TGF-β1-induced fibroblast activation were assessed by phenotypic high-content screening. Among those sixteen compounds, 1β-hydroxy alantolactone (HAL), the main characteristic sesquiterpene lactone from I. Flos, exhibited remarkable inhibitory activity. The further studies showed that HAL significantly inhibited the proliferation and induced the apoptosis of human fibroblast cell lines HELF and MRC-5 in a concentration-dependent manner. It also reduced intracellular ROS production, suppressed the mRNA expressions of E-cad, TGF-β1, Smad3, Col I, α-SMA and TNF-α, and downregulated protein expressions of α-SMA and F-actin. Furthermore, HAL significantly reduced the levels of HA, LN, PC-III and IV-C in serum, TNF-α and IL-6 in BALF, and TGF-β1, HYP and Col I in lung tissues of bleomycin (BLM)-treated rats. HAL significantly downregulated the expressions of p-JNK, FOXO1, p-p65, α-SMA, p-smad3 and Col I but upregulated p-FOXO1, which could be reversed by JNK agonist anisomycin. These results demonstrated that HAL induced the apoptosis of lung fibroblast cells activated by TGF-β1 and improved BLM-induced lung fibrosis in rats via inhibiting JNK/FOXO1/NF-κB pathway.

Curcumin inhibition of bleomycin-induced changes in lung collagen synthesis, deposition and assembly.

Idiopathic pulmonary fibrosis is characterized by progressive lung tissue remodeling and disproportionate deposition of collagenous proteins with limited therapeutic interventions. The purpose of this study was to determine whether curcumin inhibits bleomycin (BLM)-induced increases in synthesis, degradation and cross-linking of lung collagen in rats. Following a single intratracheal instillation of BLM to rats (0.75 U/100g, sacrificed 3, 5, 7, 14 and 28days post-BLM), lung collagen synthesis (determined by incorporation of 3H-proline) and deposition (determined by lung hydroxyproline content) progressively increased at days 7, 14 and 28 post-BLM injection. Lung lavage fluid hydroxyproline and collagenase levels (a measure of collagen turnover) were increased in BLM rats compared with control groups. In addition, BLM instillation resulted in increased concentrations of collagenase and collagenolytic cathepsin in the lungs. Furthermore, increased cross-linking (as determined by aldehyde content of acid soluble collagen), and decreased susceptibility of fibrotic lung insoluble collagen to denaturing agents occurred in BLM-injured lungs. Significant increases in alveolar macrophage (AM) release of transforming growth factor-β1 (TGF-β1) were noted at various time points (days 3, 5, 7, 14 and 28 post-BLM) during the development and progression of lung fibrosis in rats. Curcumin treatment to BLM rats (300mg/kg 10days before and daily thereafter throughout the experimental time period) was associated with marked reductions in lung collagen synthesis and deposition, BALF and lung collagenase activity, BALF hydroxyproline content and lung collagenolytic levels. Additionally, reduced levels of collagen cross-linking and enhanced susceptibility of insoluble lung collagen to denaturing agents were observed in curcumin-treated BLM rats. Finally, curcumin inhibited BLM-induced increases in AM production of TGF-β1. Our data demonstrate for the first time that curcumin prevents fibrotic deposits by modulating collagen turnover, assembly and deposition in BLM-instilled rat lungs, and that curcumin treatment protects against BLM activation of macrophages by suppressing the release of TGF-β1.

Combined Inhibitory Effect of Sulindac an NSAID and Quercetin an Antioxidant on Bleomycin-induced Lung Fibrosis in Rats

Combined Inhibitory Effect of Sulindac an NSAID and Quercetin an Antioxidant on Bleomycin-induced Lung Fibrosis in Rats