Qingfei Tongluo Mixture Attenuates Bleomycin-Induced Pulmonary Inflammation and Fibrosis through mTOR-Dependent Autophagy in Rats.

As an interstitial fibrosis disease characterized by diffuse alveolitis and structural alveolar disorders, idiopathic pulmonary fibrosis (IPF) has high lethality but lacks limited therapeutic drugs. A hospital preparation used for the treatment of viral pneumonia, Qingfei Tongluo mixture (QFTL), is rumored to have protective effects against inflammatory and respiratory disease. This study aims to confirm whether it has a therapeutic effect on bleomycin-induced IPF in rats and to elucidate its mechanism of action. Male SD rats were randomly divided into the following groups: control, model, CQ + QFTL (84 mg/kg chloroquine (CQ) + 3.64 g/kg QFTL), QFTL-L, M, H (3.64, 7.28, and 14.56 g/kg, respectively) and pirfenidone (PFD 420 mg/kg). After induction modeling and drug intervention, blood samples and lung tissue were collected for further detection. Body weight and lung coefficient were examined, combined with hematoxylin and eosin (H&E) and Masson staining to observe lung tissue lesions. The enzyme-linked immunosorbent assay (ELISA) and the hydroxyproline (HYP) assay kit were used to detect changes in proinflammatory factors (transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß)) and HYP. Immunohistochemistry and Western blotting were performed to observe changes in proteins related to pulmonary fibrosis (α-smooth muscle actin (α-SMA) and matrix metalloproteinase 12 (MMP12)) and autophagy (P62 and mechanistic target of rapamycin (mTOR)). Treatment with QFTL significantly improved the adverse effects of bleomycin on body weight, lung coefficient, and pathological changes. Then, QFTL reduced bleomycin-induced increases in proinflammatory mediators and HYP. The expression changes of pulmonary fibrosis and autophagy marker proteins are attenuated by QFTL. Furthermore, the autophagy inhibitor CQ significantly reversed the downward trend in HYP levels and α-SMA protein expression, which QFTL improved in BLM-induced pulmonary fibrosis rats. In conclusion, QFTL could effectively attenuate bleomycin-induced inflammation and pulmonary fibrosis through mTOR-dependent autophagy in rats. Therefore, QFTL has the potential to be an alternative treatment for IPF in clinical practice.

Qingfei xieding prescription ameliorates mitochondrial DNA-initiated inflammation in bleomycin-induced pulmonary fibrosis through activating autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingfei Xieding prescription was gradually refined and produced by Hangzhou Red Cross Hospital. The raw material includes Ephedra sinica Stapf, Morus alba L., Bombyx Batryticatus, Gypsum Fibrosum, Prunus armeniaca L. var. ansu Maxim., Houttuynia cordata Thunb. , Pueraria edulis Pamp. Paeonia L., Scutellaria baicalensis Georgi and Anemarrhena asphodeloides Bge. It is effective in clinical adjuvant treatment of patients with pulmonary diseases. AIM OF THE STUDY: To explore the efficacy and underlying mechanism of Qingfei Xieding (QF) in the treatment of bleomycin-induced mouse model. MATERIALS AND METHODS: TGF-ß induced fibrotic phenotype in vitro. Bleomycin injection induced lung tissue fibrosis mouse model in vivo. Flow cytometry was used to detect apoptosis, cellular ROS and lipid oxidation. Mitochondria substructure was observed by transmission electron microscopy. Autophagolysosome and nuclear entry of P65 were monitored by immunofluorescence. Quantitative real-time PCR was performed to detect the transcription of genes associated with mtDNA-cGAS-STING pathway and subsequent inflammatory signaling activation. RESULTS: TGF-ß induced the expression of α-SMA and Collagen I, inhibited cell viability in lung epithelial MLE-12 cells that was reversed by QF-containing serum. TGF-ß-mediated downregulation in autophagy, upregulation in lipid oxidation and ROS contents, and mitochondrial damage were rescued by QF-containing serum treatment, but CQ exposure, an autophagy inhibitor, prevented the protective role of QF. In addition to that, the decreased autophagolysosome in TGF-ß-exposed MLE-12 cells was reversed by QF and restored to low level in the combination treatment of QF and CQ. Mechanistically, QF-containing serum treatment significantly inhibited mtDNA-cGAS-STING pathway and subsequent inflammatory signaling in TGF-ß-challenged cells, which were abolished by CQ-mediated autophagy inhibition. In bleomycin-induced mouse model, QF ameliorated pulmonary fibrosis, reduced mortality, re-activated autophagy in lung tissues and restrained mtDNA-cGAS-STING inflammation pathway. However, the protective effects of QF in bleomycin-induced model mice were also abrogated by CQ. CONCLUSION: QF alleviated bleomycin-induced pulmonary fibrosis by activating autophagy, inhibiting mtDNA-cGAS-STING pathway-mediated inflammation. This research recognizes the protection role of QF on bleomycin-induced mouse model, and offers evidence for the potentiality of QF in clinical application for pulmonary fibrosis treatment.

Dangui Huoxue Preparation (DHP) Ameliorates Skin Fibrosis, Inflammation, and Vasculopathy in the Bleomycin-Induced Murine Model of Systemic Sclerosis.

Systemic sclerosis (SSc) is an immune-mediated rheumatic disease that is characterized by fibrosis of the skin and internal organs and vasculopathy with poor prognosis. Dangui Huoxue Preparation (DHP) is a clinically effective traditional Chinese herbal formula for the treatment of SSc in the hospital. This study aims to investigate the therapeutic effects and underlying molecular mechanisms of DHP in the treatment of SSc. SSc mice models are induced by bleomycin (BLM). Tissues of DHP group, normal control group, and positive control drug Sanqi Tongshu Capsule (STC) group are collected for inflammation, fibrosis, and vasculopathy. Also, the human dermal fibroblasts (HDF) stimulated with TGF-ß1 are analyzed for in vitro study. The expression levels of MCP-1, IFN-γ, IL-1ß, IL-10, Fizz1, iNOS, and IL12p40, and the mRNA levels of Col1a1, Col1a2, Col3a1, and Col5a1 are significantly decreased in all DHP groups and STC group compare with those in the BLM group. The main drug of DHP inhibits the proliferation and migration of HDF, reduces Ctgf, Itgb3, Itgb5 expression, and also inhibits the Smad3 pathway. In conclusion, DHP can ameliorate SSc skin inflammation, fibrosis, and vasculopathy, possibly suppressing the TGF-ß1/Smad3 signaling pathway through extracellular and intracellular mechanisms.

Buyang Huanwu decoction ameliorates bleomycin-induced pulmonary fibrosis in rats by attenuating the apoptosis of alveolar type II epithelial cells mediated by endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine, the pathogenesis of idiopathic pulmonary fibrosis (IPF) can be attributed to qi deficiency and blood stasis. Buyang Huanwu decoction (BHD), a representative Chinese herbal prescription for qi deficiency and blood stasis syndrome, is widely used to treat IPF in clinical practice. However, its potential mechanisms against IPF remain unclear. AIMS OF THE STUDY: This study was carried out to explore the therapeutic effects and underlying mechanisms of BHD on bleomycin (BLM)-induced pulmonary fibrosis in rats. MATERIALS AND METHODS: UPLC-MS/MS method was performed to identify the quality of BHD used in this study. Concurrently, a IPF rat model was established by single intratracheal injection of BLM. Pulmonary function test, H&E staining, Masson staining, hydroxyproline assay were conducted to evaluate the therapeutic effects of BHD on BLM-induced pulmonary fibrosis in rats, and the regulatory effect of BHD on endoplasmic reticulum stress (ERS)-mediated alveolar type II epithelial cells (AEC2s) apoptosis in rats was further investigated by TUNEL staining, Western blot, real-time fluorescence quantitative PCR and immunofluorescence co-staining to reveal the potential mechanisms of BHD against IPF. RESULTS: The UPLC-MS/MS analysis showed that the BHD we used complied with the relevant quality control standards. The data from animal experiments confirmed that BHD administration ameliorated BLM-induced pulmonary function decline, lung fibrotic pathological changes and collagen deposition in rats. Further mechanism study revealed that BHD increased the Bcl-2 protein expression, decreased the Bax protein expression and inhibited the cleavage of CASP3 via suppressing the activation of PERK-ATF4-CHOP pathway under continuous ERS, thereby alleviating BLM-induced AEC2s apoptosis of rats. CONCLUSION: This study demonstrated that BHD ameliorated BLM-induced pulmonary fibrosis in rats by suppressing ERS-mediated AEC2s apoptosis. Our findings can provide some fundamental research basis for the clinical application of BHD in the treatment of IPF.

[Aqueous extract of Epimedium sagittatum mitigates pulmonary fibrosis in mice].

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-ß1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-ß1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-ß1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.

Modeling Mediated Crossover Acute and Subacute Damage to the Pulmonary System During Systemic (Subcutaneous) Administration of Bleomycin in Mice to Test the Potential Therapeutic Effect of Polyphenols / Modelado del Daño Agudo y Subagudo Cruzado Mediado al Sistema Pulmonar Durante la Infección Sistémica (Subcutánea) de Administración de Bleomicina a Ratones para Probar el Posible Efecto Terapéutico de los Polifenoles

SUMMARY: The potential anti-inflammatory and antifibrotic activity of polyphenolic extracts of blueberry and grape was evaluated in a mouse model of lung damage induced by subcutaneous administration of bleomycin. The results of testing the polyphenolic extracts on two different systemic administration variants of bleomycin (intraperitoneal and subcutaneous) were compared. It was found that regardless of the method of bleomycin administration, indirect cross-acute and subacute damage to the pulmonary system was observed. Both patterns exhibited the same prevalence and severity. The administration of polyphenolic extracts of blueberry and grape to mice resulted in a significant decrease in theseverity of acute and subacute patterns of lung damage, suggesting their protective properties for the microcirculatory bed and a pronounced anti-inflammatory effect.

La potencial actividad antiinflamatoria y antifibrótica de los extractos polifenólicos de arándano y uva se evaluó en un modelo de daño pulmonar en ratón inducido por la administración subcutánea de bleomicina. Se compararon los resultados de las pruebas de los extractos polifenólicos en dos variantes diferentes de administración sistémica de bleomicina (intraperitoneal y subcutánea). Se encontró que, independientemente del método de administración de bleomicina, se observaba daño indirecto cruzado, agudo y subagudo al sistema pulmonar. Ambos patrones exhibieron la misma prevalencia y gravedad. La administración de extractos polifenólicos de arándano y uva a ratones dio como resultado una disminución significativa en la gravedad de los patrones agudos y subagudos de daño pulmonar, lo que sugiere sus propiedades protectoras del lecho micro- circulatorio y un efecto antiinflamatorio pronunciado.

Eupatilin inhibits pulmonary fibrosis by activating Sestrin2/PI3K/Akt/mTOR dependent autophagy pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive chronic inflammatory disease with poor clinical outcomes and ineffective drug treatment options. Eupatilin is a major component extracted from the traditional herbal medicine Artemisia asiatica Nakai. Notably, it was demonstrated to have an anti-fibrosis effect in endometrial fibrosis, vocal fold, and hepatic fibrosis. Its role and mechanism in IPF remain unclear. METHODS: This study used the TGF-ß1-induced human embryonic lung fibroblasts (MRC-5) activation, IPF lung fibroblasts, and bleomycin-induced lung fibrosis mice model. Western blot, immunofluorescence staining, quantitative real time-PCR, hematoxylin and eosin staining, Masson's trichrome staining, and immunohistochemistry were used to evaluate the effects of eupatilin on fibroblast activation, pulmonary fibrosis, and autophagy. The autophagosomes were observed with a transmission electron microscope (TEM). RNA sequencing was used to determine the signaling pathway and key regulator related to autophagy. RESULTS: Eupatilin significantly decreased the expression of Col1A1, fibronectin, α-SMA, and SQSTM1/p62. In contrast, it increased the expression of LC3B II/I and the number of autophagosomes in TGF-ß1 treated MRC-5, IPF lung fibroblasts, and bleomycin-induced lung fibrosis mice model; it also alleviated bleomycin-induced lung fibrosis. The KEGG pathway mapping displayed that PI3K/Akt and Sestrin2 were associated with the enhanced fibrogenic process. Eupatilin suppressed the phosphorylation of PI3K/Akt/mTOR. Autophagy inhibitor 3-methyladenine (3-MA) and Akt activator SC-79 abrogated the anti-fibrotic effect of eupatilin. Sestrin2 expression was also downregulated in TGF-ß1 treated lung fibroblasts and lung tissues of the bleomycin-induced pulmonary fibrosis mice model. Furthermore, eupatilin promoted Sestrin2 expression, and the knockdown of Sestrin2 significantly aggravated the degree of fibrosis, increased the phosphorylation of PI3K/Akt/mTOR, and decreased autophagy. CONCLUSION: These findings indicate that eupatilin ameliorates pulmonary fibrosis through Sestrin2/PI3K/Akt/mTOR-dependent autophagy pathway.

Icariside â ¡ attenuates bleomycin-induced pulmonary fibrosis by modulating macrophage polarization.

ETHNOPHARMACOLOGICAL RELEVANCE: Numerous studies have provided evidence supporting the significant roles of icariin, in the prevention of multiple chronic diseases like diabetes, liver fibrosis, cardiac fibrosis, renal fibrosis, and pulmonary fibrosis. In particular, Icariside II (ISE II), a prominent flavonoid glycoside derived from Epimedium brevicornum Maxim, the principal metabolite of icariin, has demonstrated noteworthy anti-inflammatory and anti-oxidant properties, along with its ability to protect against lung remodeling. However, the research exploring ISE Ⅱ's application in treating pulmonary fibrosis remains limited. AIM OF THE STUDY: The aim of this study was to assess the therapeutic efficacy of ISE II in models of pulmonary fibrosis, while also investigating its potential mechanisms of action in cell signaling pathways. MATERIALS AND METHODS: An in vitro model of pulmonary fibrosis was established by treating NIH-3T3 cells with transforming growth factor-ß1 (TGF-ß1). Western blot, RT-qPCR, and scratch test were performed to assess the effect of ISE Ⅱ. In addition, a murine model of pulmonary fibrosis was induced by intratracheal instillation of bleomycin, and the therapeutic effect of ISE Ⅱ was tested by orally administering ISE Ⅱ at a dose of 10 mg/kg. Three weeks later, lung function, micro-CT, hydroxyproline content, pathological staining, and cytokines detection of BALF or serum were used to assess the anti-fibrosis effects of ISE Ⅱ. Next, immunofluorescence staining, flow cytometry, and in vivo transcriptomics were used to investigate the underlying mechanisms of action. RESULTS: Our data revealed a significant inhibitory effect of ISE Ⅱ on the upregulation of α-smooth muscle actin (α-SMA) and collagen production induced by TGF-ß1 in fibroblasts. Meanwhile, ISE Ⅱ exerted a therapeutic effect against bleomycin-induced pulmonary fibrosis in mice by improving lung function, decreasing collagen deposition, and reducing the expression of interleukin (IL)-1ß, tumor necrosis factor α (TNF-α), TGF-ß1 and platelet-derived growth factor (PDGF) in serum and bronchoalveolar lavage fluid (BALF). Additionally, ISE Ⅱ treatment effectively attenuated the infiltration of M2 macrophages, concurrently downregulating the expression level of M2 marker genes, such as CD206, arginase-1(Arg-1), and Chitinase-Like Protein 3 (YM-1). Importantly, we observed a statistically significant reduction in the M2 phenotype of interstitial macrophages (IMs). However, the impact of ISE Ⅱ on the M2 polarization of alveolar macrophages (AMs) did not reach statistical significance. Lastly, transcriptome sequencing results suggested that the anti-pulmonary fibrosis effects of ISE Ⅱ may be mediated by the suppression of the WNT/ß-catenin signaling pathway, which modulated M2 polarization in macrophages and contributed to the amelioration of pulmonary fibrosis. By immunohistochemical analysis, it was verified that ISE Ⅱ treatment dramatically inhibited the activation of ß-catenin in fibrosis murine. CONCLUSION: Our findings indicated that ISE Ⅱ exerted anti-fibrotic effects by inhibiting pro-fibrotic macrophage polarization. The underlying mechanism of action might be mediated by modulating the WNT/ß-catenin signaling pathway to inhibit the M2 program in IMs.

Arginine concentration in arterial vs venous blood in a bleomycin-induced lung inflammation model in mice.

Pneumonia, always a major malady, became the main public health and economic disaster of historical proportions with the COVID-19 pandemic. This study was based on a premise that pathology of lung metabolism in inflammation may have features invariant to the nature of the underlying cause. Amino acid uptake by the lungs was measured from plasma samples collected pre-terminally from a carotid artery and vena cava in mice with bleomycin-induced lung inflammation (N = 10) and compared to controls treated with saline instillation (N = 6). In the control group, the difference in concentrations between the arterial and venous blood of the 19 amino acids measured reached the level of statistical significance only for arginine (-10.7%, p = 0.0372) and phenylalanine (+5.5%, p = 0.0266). In the bleomycin group, 11 amino acids had significantly lower concentrations in the arterial blood. Arginine concentration was decreased by 21.1% (p<0.0001) and only that of citrulline was significantly increased (by 20.1%, p = 0.0002). Global Arginine Bioavailability Ratio was decreased in arterial blood by 19.5% (p = 0.0305) in the saline group and by 30.4% (p<0.0001) in the bleomycin group. Production of nitric oxide (NO) and citrulline from arginine by the inducible nitric oxide synthase (iNOS) is greatly increased in the immune system's response to lung injury. Deprived of arginine, the endothelial cells downstream may fail to provide enough NO to prevent the activation of thrombocytes. Thrombotic-related vascular dysfunction is a defining characteristic of pneumonia, including COVID-19. This experiment lends further support to arginine replacement as adjuvant therapy in pneumonia.

Inositol possesses antifibrotic activity and mitigates pulmonary fibrosis.

BACKGROUND: Myo-inositol (or inositol) and its derivatives not only function as important metabolites for multiple cellular processes but also act as co-factors and second messengers in signaling pathways. Although inositol supplementation has been widely studied in various clinical trials, little is known about its effect on idiopathic pulmonary fibrosis (IPF). Recent studies have demonstrated that IPF lung fibroblasts display arginine dependency due to loss of argininosuccinate synthase 1 (ASS1). However, the metabolic mechanisms underlying ASS1 deficiency and its functional consequence in fibrogenic processes are yet to be elucidated. METHODS: Metabolites extracted from primary lung fibroblasts with different ASS1 status were subjected to untargeted metabolomics analysis. An association of ASS1 deficiency with inositol and its signaling in lung fibroblasts was assessed using molecular biology assays. The therapeutic potential of inositol supplementation in fibroblast phenotypes and lung fibrosis was evaluated in cell-based studies and a bleomycin animal model, respectively. RESULTS: Our metabolomics studies showed that ASS1-deficient lung fibroblasts derived from IPF patients had significantly altered inositol phosphate metabolism. We observed that decreased inositol-4-monophosphate abundance and increased inositol abundance were associated with ASS1 expression in fibroblasts. Furthermore, genetic knockdown of ASS1 expression in primary normal lung fibroblasts led to the activation of inositol-mediated signalosomes, including EGFR and PKC signaling. Treatment with inositol significantly downregulated ASS1 deficiency-mediated signaling pathways and reduced cell invasiveness in IPF lung fibroblasts. Notably, inositol supplementation also mitigated bleomycin-induced fibrotic lesions and collagen deposition in mice. CONCLUSION: These findings taken together demonstrate a novel function of inositol in fibrometabolism and pulmonary fibrosis. Our study provides new evidence for the antifibrotic activity of this metabolite and suggests that inositol supplementation may be a promising therapeutic strategy for IPF.

Derrone Targeting the TGF Type 1 Receptor Kinase Improves Bleomycin-Mediated Pulmonary Fibrosis through Inhibition of Smad Signaling Pathway.

Transforming growth factor-ß (TGF-ß) has a strong impact on the pathogenesis of pulmonary fibrosis. Therefore, in this study, we investigated whether derrone promotes anti-fibrotic effects on TGF-ß1-stimulated MRC-5 lung fibroblast cells and bleomycin-induced lung fibrosis. Long-term treatment with high concentrations of derrone increased the cytotoxicity of MRC-5 cells; however, substantial cell death was not observed at low concentrations of derrone (below 0.05 µg/mL) during a three-day treatment. In addition, derrone significantly decreased the expressions of TGF-ß1, fibronectin, elastin, and collagen1α1, and these decreases were accompanied by downregulation of α-SMA expression in TGF-ß1-stimulated MRC-5 cells. Severe fibrotic histopathological changes in infiltration, alveolar congestion, and alveolar wall thickness were observed in bleomycin-treated mice; however, derrone supplementation significantly reduced these histological deformations. In addition, intratracheal administration of bleomycin resulted in lung collagen accumulation and high expression of α-SMA and fibrotic genes-including TGF-ß1, fibronectin, elastin, and collagen1α1-in the lungs. However, fibrotic severity in intranasal derrone-administrated mice was significantly less than that of bleomycin-administered mice. Molecular docking predicted that derrone potently fits into the ATP-binding pocket of the TGF-ß receptor type 1 kinase domain with stronger binding scores than ATP. Additionally, derrone inhibited TGF-ß1-induced phosphorylation and nuclear translocations of Smad2/3. Overall, derrone significantly attenuated TGF-ß1-stimulated lung inflammation in vitro and bleomycin-induced lung fibrosis in a murine model, indicating that derrone may be a promising candidate for preventing pulmonary fibrosis.

Jingfang Granule alleviates bleomycin-induced acute lung injury via CD200-CD200R immunoregulatory pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jingfang granules (JF), one famous traditional Chinese formula in "She Sheng Zhong Miao Fang" written by Shi-Che Zhang during the Ming Dynasty era, has been widely used to prevent epidemic diseases in history and now was recommended for the treatment of coronavirus disease 2019 (COVID-19) in China. However, the roles of JF against acute lung injury and its mechanisms remain unclear. AIM OF THE STUDY: Acute lung injury (ALI) and its progressive acute respiratory distress syndrome (ARDS) are a continuum of lung inflammatory disease with high morbidity and mortality in clinic, especially in COVID-19 patients. The present study aims to investigate the effect of JF on ALI and clarify its underlying mechanisms for clinical application in COVID-19 control. METHODS: Bleomycin-induced ALI mice were given oral gavage daily for seven days with or without Jingfang granules (2, 4 g/kg). The body weight, lung wet/dry weight ratios, lung appearance and tissue histopathology were evaluated. Quantitative real-time PCR, biochemical bronchoalveolar lavage fluids analysis was used to determine the gene expression of proinflammation factor and infiltrated inflammatory cells in lung. Immunofluorescence image and western blot were used to detect the markers of alveolar macrophages (AMs), endothelial cell apoptosis and changes of CD200-CD200R pathway. RESULTS: Firstly, histopathological analysis showed that JF significantly attenuated pulmonary injury and inflammatory response in ALI mice. Then, cytokine detection, inflammatory cells assay, and JNKs and p38 pathway analysis indicated that the recruitment and activation of alveolar macrophages was the main reason to cause ALI and JF could reverse this variation. Next, immunofluorescence staining and TUNEL assay showed that JF upregulated the expression of CD200 and suppressed the apoptosis of alveolar endothelial cells. Finally, double immunofluorescence staining of CD200 and CD11c indicated that the seriously damaged tissue had the lower CD200 while more AMs infiltration, which was confirmed by RT-PCR analysis of CD200/CD200R. CONCLUSIONS: Jingfang granules can protect lung from acu te injury and mitigate the recruitment and overactive AMs-induced inflammation via CD200-CD200R immunoregulatory signal axis, which will provide an experimental basis for Jingfang granules clinical applications in COVID-19.

Simiao pill attenuates collagen-induced arthritis and bleomycin-induced pulmonary fibrosis in mice by suppressing the JAK2/STAT3 and TGF-ß/Smad2/3 signalling pathway.

ETHNOPHAMACOLOGICAL RELEVANCE: Simiao Pill (SM) as a classic prescription of traditional Chinese medicine treatment of damp-heat arthralgia, the earliest from 'Cheng Fan Bian Du ', written by the Qing Dynasty doctor Zhang Bingcheng. Previous studies have shown that SM has obvious curative effect on rheumatoid arthritis, which provides a basis for the application of SM in rheumatoid arthritis related complications. AIM OF THE STUDY: Interstitial lung disease (ILD), as the most severe complication of rheumatoid arthritis (RA), lacks effective clinical treatments and a corresponding animal model. Simiao pill (SM) is a traditional Chinese medicine prescription extensively used as a complementary and alternative treatment for RA. However, the effect and mechanism of SM on RA-ILD have not yet been reported. This study aimed to investigate an appropriate animal model that can simulate RA-ILD, and the efficacy, safety, and mechanism of SM on RA-ILD. METHODS: Collagen-induced arthritis (CIA) and bleomycin-induced pulmonary fibrosis model were combined to construct the CIA-BLM model. After the intervention of SM, the protective effects of SM on RA-ILD were determined by detecting the CIA mouse arthritis index (AI), Spleen index, and the extent of pulmonary fibrosis. The joint inflammation and pulmonary fibrosis were detected by immunohistochemistry, H&E staining, safranin- O fast green Sirius red staining, trap staining, and Masson staining. Finally, the mechanism was verified by Western blot and immunohistochemistry. RESULTS: Our work showed that SM significantly reduced joint swelling, arthritis index, pulmonary fibrosis score, and spleen index in CIA mice. The pathological examination results indicated Si-Miao Pill suppressed inflammation, pulmonary fibrosis, bone erosion, and cartilage degradation of the ankle joint. Besides, SM up-regulated expressions of E-cadherin, whereas down-regulated expressions of α-SMA. Further studies confirmed that SM regulated JAK2/STAT3 and TGF-ß/SMAD2/3. CONCLUSION: SM can not only effectively improve joint inflammation by JAK2/STAT3 Pathway but also inhibit pulmonary fibrosis by TGF-ß/SMAD2/3. The fibrosis induced by CIA-BLM model was more stable and obvious than that induced by CIA model alone.

Qimai Feiluoping decoction inhibits mitochondrial complex I-mediated oxidative stress to ameliorate bleomycin-induced pulmonary fibrosis.

BACKGROUND: Qimai Feiluoping decoction (QM), a Traditional Chinese Medicine formula, has been included in rehabilitation program for functional disorders of discharged COVID-19 patients. QM has been proved to effectively improve the clinical symptoms and imaging signs of PF in COVID-19 convalescent patients. PURPOSE: This study to explore the pharmacological effect of QM against PF from the perspectives of imaging, pathological staining, and molecular mechanisms, and identify possible active components. METHODS: Micro-CT imaging and immunohistochemical staining were investigated to verify the therapeutic effect of QM in the bleomycin (BLM)-induced PF mouse model. The 4D-label-free proteomics analysis of lung tissues was then conducted to explore the novel mechanisms of QM against PF, which were further validated by a series of experiments. The possible components of QM in plasma and lung tissues were identified with UHPLC/IM-QTOF-MS analysis. RESULTS: The results from micro-CT imaging and pathological staining revealed that QM treatment can inhibit BLM-induced lung injury, extracellular matrix accumulation and TGF-ß expression in the mouse model with PF. The 4D-label-free proteomics analysis demonstrated that the partial subunit proteins of mitochondrial complex I and complex II might be potential targets of QM against PF. Furthermore, QM treatment can inhibit BLM-induced mitochondrial ROS content to promote ATP production and decrease oxidative stress injury in the mouse and cell models of PF, which was mediated by the inhibition of mitochondrial complex I. Finally, a total of 13 protype compounds and 15 metabolites from QM in plasma and lung tissues were identified by UHPLC/IM-QTOF-MS, and liquiritin and isoliquiritigenin from Glycyrrhizae radix et rhizoma could be possible active compounds against PF. CONCLUSION: It concludes that QM treatment could treat PF by inhibiting mitochondrial complex I-mediated mitochondrial oxidated stress injury, which could offer new insights into the pharmacological mechanisms of QM in the clinical application of PF patients.

Potential antifibrotic effect of blueberry and grape polyphenol extracts in acute and subacute bleomycin-induced lung tissue injury models in mice / Potencial efecto antifibrótico de los extractos de polifenoles de arándano y uva en modelos de lesión tisular pulmonar aguda y subaguda inducida por bleomicina en ratones

SUMMARY: An experimental morphological and morphometric study of the antifibrotic function of blueberry and grape extracts was carried out on a model of lung injury in mice induced by intraperitoneal administration of bleomycin. During intraperitoneal administration of bleomycin to mice, acute and subacute damage to the pulmonary system was noted. Both patterns had the same prevalence and severity. The administration of polyphenolic extracts of blueberry and grape to mice showed a significant reduction in the severity of the acute and subacute pattern of lung injury. Blueberry and grape extracts reduce the acute phase of damage to the microvasculature, enhance phagocytic function, have an anti-inflammatory effect, reducing the degree of lymphohistiocytic infiltration and locoregional foci of residual inflammatory effects.

Se realizó un estudio experimental morfológico y morfométrico de la función antifibrótica de extractos de arándano y uva en un modelo de lesión pulmonar en ratones inducida por la administración intraperitoneal de bleomicina. Durante la administración intraperitoneal de bleomicina a ratones, se observaron daños agudos y subagudos en el sistema pulmonar. Ambos patrones tuvieron la misma prevalencia y severidad. La administración de extractos polifenólicos de arándano y uva a ratones mostró una reducción significativa en la severidad del patrón agudo y subagudo de lesión pulmonar. Los extractos de arándano y uva reducen la fase aguda del daño a la microvasculatura, mejoran la función fagocítica, tienen un efecto antiinflamatorio, reducen el grado de infiltración linfohistiocítica y los focos locorregionales de efectos inflamatorios residuales.

Tanreqing injection protects against bleomycin-induced pulmonary fibrosis via inhibiting STING-mediated endoplasmic reticulum stress signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Idiopathic pulmonary fibrosis (IPF), characterized by excessive collagen deposition, is a progressive and typically fatal lung disease without effective therapeutic methods. Tanreqing injection (TRQ), a Traditional Chinese Patent Medicine, has been widely used to treat inflammatory respiratory diseases clinically. AIM OF THE STUDY: The present work aims to elucidate the therapeutic effects and the possible mechanism of TRQ against pulmonary fibrosis. METHODS: The pulmonary fibrosis murine model were constructed by the intratracheal injection of bleomycin (BLM). 7 days later, TRQ-L (2.6 ml/kg) and TRQ-H (5.2 ml/kg) were administered via intraperitoneal injection respectively for 21 days. The efficacy and underlying molecular mechanism of TRQ were investigated. RESULTS: Here, we showed that TRQ significantly inhibited BLM-induced lung edema and pulmonary function. TRQ markedly reduced BLM-promoted inflammatory cell infiltration in BALF and inflammatory cytokines release (TNF-α, IL-6, and IL-1ß) in serum and lung tissues. Meanwhile, TRQ also alleviated BLM-induced collagen synthesis and deposition. Simultaneously, TRQ attenuated BLM-induced pulmonary fibrosis through regulating the expression of fibrotic hallmarks, manifested by down-regulated α-SMA and up-regulated E-cadherin. Moreover, we found that TRQ significantly prevented STING, p-P65, BIP, p-PERK, p-eIF2α, and ATF4 expression in lung fibrosis mice. CONCLUSIONS: Taken together, our results indicated that TRQ positively affects inflammatory responses and lung fibrosis by regulating STING-mediated endoplasmic reticulum stress (ERS) signal pathway.

Protective Effect of Combined Moxibustion and Decoction Therapy on Bleomycin-Induced Pulmonary Fibrosis in Rats under Nuclear Factor-κB/Transforming Growth Factor-ß1/Smads Signaling Pathway.

it was aimed to discuss the effect of moxibustion (Mox) combined with Bu Fei Qu Yu (BFQY) decoction under the nuclear factor-κB (NF-κB)/transforming growth factor-ß1 (TGF-ß1)/Smads signaling pathway in the treatment of pulmonary fibrosis (PF). The PF rat models were prepared with bleomycin (BLM). They were divided into the normal (Nor) group, the PF model group (BLM puncture perfusion), the Mox group (grain-sized Mox at the back-shu points and Xuxiao points), the BFQY group (intragastrical BFQY decoction), and the Mox combined with BFQY decoction (Mox+BFQY) group. Lung tissue sections were prepared, and the hematoxylin-eosin (HE) staining and Masson staining were performed to observe the inflammatory response and the degree of PF. The contents of hydroxyproline (HYP), glutathione (GSH), and malondialdehyde (MDA), and the expressions of NF-κB p65, TGF-ß1, Smad2, and Smad7 in lung tissues were detected. Compared with those in the Nor group, the inflammatory response score, PF degree score, HYP, GSH, and MDA contents, NF-κB p65, TGF-ß1, and Smad2 expressions were significantly increased in the PF group, but Smad7 expression decreased (P<0.05). The above symptoms were significantly improved in the Mox, BFQY, and Mox+ BFQY groups (P<0.05). The effect was more remarkable in the Mox+BFQY group, and there was no significant difference in each index compared with those in the Nor group (P>0.05). Thus, the combined therapy of Mox and decoction had an effect on PF through the NF-κB/TGF-ß1/Smads pathway.

Aprepitant exerts anti-fibrotic effect via inhibition of TGF-ß/Smad3 pathway in bleomycin-induced pulmonary fibrosis in rats.

Bleomycin is a well-recognized antineoplastic drug. However, pulmonary fibrosis (PF) is considered to be the principal drawback that greatly limits its use. Here, we sought to investigate ability of the neurokinin receptor 1 blocker, aprepitant, to prevent PF caused by bleomycin. Male adult Wistar rat groups were given a single intratracheal injection of bleomycin, either alone or in combination with aprepitant therapy for 3 or 14 days. Collagen deposition and a rise in transforming growth factor beta (TGF-ß) immunoreactivity in lung tissue serve as evidence of bleomycin-induced PF. The serum levels of lactate dehydrogenase, alkaline phosphatase, and total antioxidant improved after aprepitant therapy.Additionally, it reduced the protein expressions of interferon alpha, tumor necrosis factor alpha, and lung lipid peroxidation. Moreover, aprepitant treatment led to an increase in the antioxidant indices glutathione, glutathione peroxidase, and catalase. Aprepitant is postulated to protect against bleomycin-induced PF by decreasing TGF-ß, phosphorylating Smad3, and increasing interleukin 37, an anti-fibrotic cytokine, and G Protein-coupled Receptor Kinase 2. Aprepitant for 14 days considerably exceeded aprepitant for 3 days in terms of improving lung damage and having an anti-fibrotic impact. In conclusion, aprepitant treatment for 14 days may be used as an adjuvant to bleomycin therapy to prevent PF, mostly through inhibiting the TGF-/p-Smad3 fibrotic pathway.

GSPE Protects against Bleomycin-Induced Pulmonary Fibrosis in Mice via Ameliorating Epithelial Apoptosis through Inhibition of Oxidative Stress.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown cause which leads to alveolar epithelial cell apoptosis followed by basement membrane disruption and accumulation of extracellular matrix, destroying the lung architecture. Oxidative stress is involved in the development of alveolar injury, inflammation, and fibrosis. Oxidative stress-mediated alveolar epithelial cell (AEC) apoptosis is suggested to be a key process in the pathogenesis of IPF. Therefore, the present study investigated whether grape seed proanthocyanidin extract (GSPE) could inhibit the development of pulmonary fibrosis via ameliorating epithelial apoptosis through the inhibition of oxidative stress. We found that GSPE significantly ameliorated the histological changes and the level of collagen deposition in bleomycin (BLM)-induced lungs. Moreover, GSPE attenuated lung inflammation by reducing the total number of cells in bronchoalveolar lavage (BAL) fluid and decreasing the expression of IL-6. We observed that the levels of H2O2 leading to oxidative stress were increased following BLM instillation, which significantly decreased with GSPE treatment both in vivo and in vitro. These findings showed that GSPE attenuated BLM-induced epithelial apoptosis in the mouse lung and A549 alveolar epithelial cell through the inhibition of oxidative stress. Furthermore, GSPE could attenuate mitochondrial-associated cell apoptosis via decreasing the Bax/Bcl-2 ratio. The present study demonstrates that GSPE could ameliorate bleomycin-induced pulmonary fibrosis in mice via inhibition of epithelial apoptosis through the inhibition of oxidative stress.

[Mechanism of total flavonoid extract from Dracocephalum moldavica on bleomycin-induced pulmonary fibrosis in mice based on pyroptosis pathway].

This study investigated the mechanism of total flavonoid extract from Dracocephalum moldavica(TFDM) in mice with bleomycin(BLM)-induced pulmonary fibrosis(PF) and explored its mechanism against the pyroptosis pathway. A mouse model of PF was established by intratracheal infusion of bleomycin(4 mg·kg~(-1)), and the normal group was treated with the same dose of saline under the same conditions. After the second day of modeling, the distilled water was given to the normal and model groups by gavage, and the corresponding drug were given to the TFDM and the dexamethasone groups for 28 consecutive days. After 28 days, lung tissues of mice with PF were taken to determine the content of hydroxyproline(HYP). The degree of lung inflammation and fibrosis was observed by hematoxylin-eosin(HE) and Masson stainings, and the content of interleukin-18(IL-18) and interleukin-1ß(IL-1ß) in the serum of mice with PF were measured by enzyme-linked immunosorbent assay(ELISA). Western blot was used to determine the expression levels of proteins in the lung tissues of mice with PF. HE staining showed that the BLM group had abnormal lung tissue structures and showed more inflammatory cell infiltration. Masson staining showed plenty of collagenous fibrotic tissues that were stained blue in the lung tissues. As compared with the normal group, the content of HYP and levels of IL-18 and IL-1ß in the serum of rats in the BLM group were up-regulated(P<0.01). The protein expressions of type Ⅰ collagen(Col-1), fibronectin 1(FN1), α-smooth muscle actin(α-SMA), cysteinyl aspartate specific proteinase-1(caspase-1), gasdermin D(GSDMD), NOD-like receptor thermal protein domain associated protein 3(NLRP3), p62, and apoptosis-associated speck-like protein containing a CARD(ASC) in the lung tissues of mice with PF in the BLM group were increased(P<0.01), whereas the protein expressions of autophagy-related 5(ATG5) and Beclin1 were decreased(P<0.01). Compared with the BLM group, the TFDM groups and dexamethasone group showed normal lung tissue structures and reduced inflammatory cell infiltration. Less collagenous fibrous tissues in blue color were seen and the fibrosis in the lung tissue was alleviated in the TFDM groups and dexamethasone group, with the down-regulation of the content of HYP and the levels of IL-18 and IL-1ß(P<0.05, P<0.01). In the TFDM groups and dexamethasone group, the protein expression levels of Col-1, FN1, α-SMA, caspase-1, GSDMD, NLRP3, p62, and ASC were decreased(P<0.01), and the protein expressions of ATG5 and Beclin1 were increased(P<0.01) in the lung tissues of mice with PF. From the above results, it is known that TFDM down-regulates the levels of inflammatory factors and related proteins, and effectively mitigates the process of BLM-induced PF by regulating the pyroptosis pathways and potentially affecting the autophagy.