Lung Tissue Protein Research Articles

MPO interacts with hRSV particles, contributing to the virucidal effects of NETs against clinical and laboratory hRSV isolates

Human Respiratory syncytial virus (hRSV) mainly affects immunosuppressed patients requiring hospitalization. No specific treatment is financially accessible, and available vaccines do not cover all risk groups. During hRSV infection, there is a robust neutrophilic influx into the airways. hRSV-activated neutrophils release substantial neutrophil extracellular traps (NETs) in lung tissue, comprising DNA, histones, cytosolic, and granular proteins. NETs form mucus buildup in the lungs, compromising respiratory capacity and neutralizing viral particles. Understanding responsible NETs molecules requires improvement. We evaluated NETs interacting with hRSV particles and their contribution to anti-hRSV NET effects. Immunoblotting, immunoprecipitation, and peptide sequencing assays confirmed hRSV binding to a 50–75 kDa NET protein, Myeloperoxidase (MPO). MPO, a microbicide enzyme in NETs, interacts with hRSV, likely at F0 protein (site IV) on the viral surface. Additionally, MPO (32 μM) and NETs (0.4 μg/mL) reduced in vitro replication of clinical (hRSV A and B) and laboratory (Long) hRSV isolates by approximately 30 %, reversible by selective MPO inhibitor (PF-06281355; 48 μM). Thus, MPO contributes to virucidal NET effects on diverse hRSV strains, enhancing comprehension of NETs' role in infection and aiding treatment strategies for respiratory diseases.

Sodium Hydrosulfide Protects Rats from Hypobaric-Hypoxia-Induced Acute Lung Injury

Hydrogen sulfide (H2S), as a key gas signaling molecule, plays an important role in regulating various diseases, with appropriate concentrations providing antioxidative, anti-inflammatory, and anti-apoptotic effects. The specific role of H2S in acute hypoxic injury remains to be clarified. This study focuses on the H2S donor sodium hydrosulfide (NaHS) and explores its protective effects and mechanisms against acute hypoxic lung injury. First, various mouse hypoxia models were established to evaluate H2S’s protection in hypoxia tolerance. Next, a rat model of acute lung injury (ALI) induced by hypoxia at 6500 m above sea level for 72 h was created to assess H2S’s protective effects and mechanisms. Evaluation metrics included blood gas analysis, blood routine indicators, lung water content, and lung tissue pathology. Additionally, LC-MS/MS and bioinformatic analyses were combined in performing quantitative proteomics on lung tissues from the normoxic control group, the hypoxia model group, and the hypoxia model group with NaHS treatment to preliminarily explore the protective mechanisms of H2S. Further, enzyme-linked immunosorbent assays (ELISA) were used to measure oxidative stress markers and inflammatory factors in rat lung tissues. Lastly, Western blot analysis was performed to detect Nrf2, HO-1, P-NF-κB, NF-κB, HIF-1α, Bcl-2, and Bax proteins in lung tissues. Results showed that H2S exhibited significant anti-hypoxic effects in various hypoxia models, effectively modulating blood gas and blood routine indicators in ALI rats, reducing pulmonary edema, improving lung tissue pathology, and alleviating oxidative stress, inflammatory responses, and apoptosis levels.

Schisandrol A Alleviates Allergic Asthma in Mice via Regulating the NF-κB/IκBα and Nrf2/HO-1 Signaling Pathways.

Schisandra chinensis (Turcz) Baill (S. chinensis) is the key traditional Chinese medicine for the treatment of asthma used by ancient and modern medical practitioners. However, the material basis and the main mechanism of its antiasthmatic effect remain unclear. Our preliminary results showed that schisandrol A (SCA), a representative monomer of Schisandra lignans, had the best relaxation effect on tracheal rings in isolated rats. In this research, a mouse asthma model was prepared by combining ovalbumin (OVA) with Al (OH)3 for exploring the antiasthmatic action and the underlying mechanism of SCA. The study results demonstrated that SCA improved the behavior of mice with asthma and pathological changes in their lung tissues and airways, decreased serum immunoglobulin E (IgE) and OVA-IgE levels, interleukin-4 (IL-4), IL-5, IL-13, and eotaxin contents, and leukocytes number in bronchoalveolar lavage fluid. SCA downregulated the gene expressions of keratinocyte-derived protein chemokines and ILs and reduced the expressions of phosphorylated IκB kinase α (p-IKKα) and p-nuclear factor kappa-B (NF-κB) proteins in lung tissues. In addition, it was found that SCA could significantly increase T-superoxide dismutase and catalase activities, decrease malondialdehyde content, and elevate p-IκBα, NF-E2-related-factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein expressions. In summary, SCA treatment resulted in a significant improvement in the allergic bronchial asthma in mice, and its mechanisms may involve the regulation of the NF-κB/IκBα pathway to reduce inflammatory response and the Nrf2/HO-1 pathway to improve the body's antioxidant capacity. These results suggest that SCA is a key component of S. chinensis in exerting antiasthmatic effects.

Dachengqi decoction dispensing granule ameliorates LPS-induced acute lung injury by inhibiting PANoptosis in vivo and in vitro

Ethnopharmacological relevanceAcute lung injury (ALI) is a serious health-threatening syndrome of intense inflammatory response in the lungs, with progression leading to acute respiratory distress syndrome (ARDS). Dachengqi decoction dispensing granule (DDG) has a pulmonary protective role, but its potential modulatory mechanism to alleviate ALI needs further excavation. Aim of the studyThis study aims to investigate the effect and potential mechanism of DDG on lipopolysaccharide (LPS)-induced ALI models in vivo and in vitro. Materials and methodsLPS-treated Balb/c mice and BEAS-2B cells were used to construct in vivo and in vitro ALI models, respectively. Hematoxylin-eosin (HE), Wet weight/Dry weight (W/D) calculation of lung tissue, and total protein and Lactic dehydrogenase (LDH) assays in BALF were performed to assess the extent of lung tissue injury and pulmonary edema. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in BALF, serum, and cell supernatant. The qRT-PCR was used to detect inflammatory factors, Z-DNA binding protein 1 (ZBP1), and receptor-interacting protein kinase 1 (RIPK1) expression in lung tissues and BEAS-2B cells. Double immunofluorescence staining and co-immunoprecipitation were used to detect the relative expression and co-localization of ZBP1 and RIPK1. The effects of LPS and DDG on BEAS-2B cell activity were detected by Cell Counting Kit-8 (CCK-8). Western blot (WB) was performed to analyze the expression of PANoptosis-related proteins in lung tissues and BEAS-2B cells. ResultsIn vivo, DDG pretreatment could dose-dependently improve the pathological changes of lung tissue in ALI mice, and reduce the W/D ratio of lung, total protein concentration, and LDH content in BALF. In vitro, DDG reversed the inhibitory effect of LPS on BEAS-2B cell viability. Meanwhile, DDG significantly reduced the levels of inflammatory factors in vitro and in vivo. In addition, DDG could inhibit the expression levels of PANoptosis-related proteins, especially the upstream key regulatory molecules ZBP1 and RIPK1. ConclusionDDG could inhibit excessive inflammation and PANoptosis to alleviate LPS-induced ALI, thus possessing good anti-inflammatory and lung-protective effects. This study establishes a theoretical basis for the further development of DDG and provides a new prospect for ALI treatment by targeting PANoptosis.

Ultrafiltration of Angelicae Sinensis Radix and Astragali Radix inhibits ferroptosis and improves pulmonary fibrosis in rats by regulating Nrf2/xCT/GPX4 signaling pathway

This study aims to investigate the mechanism of ferroptosis mediated by the nuclear factor-E2-related factor 2(Nrf2)/solute carrier family 7 member 11(SLC7A11, also known as xCT)/glutathione peroxidase 4(GPX4) signaling pathway in radiationinduced pulmonary fibrosis and the intervention effect of Angelicae Sinensis Radix(ASR) and Astragali Radix(AR) ultrafiltration extract. Fifty Wistar rats were randomly divided into five groups, with 10 rats in each group. Except for the blank group without radiation, the rats in each group were anesthetized and subjected to a single local chest irradiation of 40 Gy X-rays once to establish a rat model of radiation-induced pulmonary fibrosis. After radiation, the rats in the intervention groups were orally administered with ASR-AR ultrafiltration extract at doses of 0. 12, 0. 24, and 0. 48 g·kg~(-1), respectively, once a day for 30 days. After 30 days of continuous administration, the levels of oxidative stress indicators superoxide dismutase(SOD) activity, reduced glutathione(GSH),malondialdehyde(MDA), and ferrous ion(Fe~(2+)) in lung tissues of each group were detected by colorimetry. Immunofluorescence was used to detect reactive oxygen species(ROS) fluorescence expression in lung tissues. Hematoxylin-eosin(HE) and Masson staining were performed to observe pathological changes in lung tissues. Immunohistochemistry and Western blot were used to detect the expression levels of Nrf2/xCT/GPX4 signaling pathway and fibrotic proteins in lung tissues. The results showed that compared with the results in the blank group, the levels of Fe~(2+) and MDA in the model group increased, while SOD activity and GSH levels decreased,and ROS levels increased. HE and Masson staining results showed that the structure of lung tissue was seriously damaged, the pulmonary interstitium was significantly proliferated, the alveoli collapsed and consolidated severely, and there were more inflammatory cell aggregates and collagen fiber deposits. Transmission electron microscopy showed that the degree of lung tissue damage in the model group was relatively high, with increased, smaller, and disorganized damaged mitochondria, irregular morphology, shallow matrix,most mitochondria ruptured and shortened, mildly expanded, some mitochondria with increased electron density of the matrix, partial mitochondrial outer membrane rupture, and characteristic changes of ferroptosis-specific mitochondria. Immunohistochemistry showed that the expression of transferrin receptor protein 1(TFR1) in lung tissues was significantly increased, while the expression of GPX4,ferritin heavy chain 1(FTH1), Nrf2, and xCT was significantly decreased. Western blot showed that the expression of α-smooth muscle actin(α-SMA) and collagen Ⅰ protein increased. Compared with the model group, the intervention group with ASR-AR ultrafiltration extract significantly improved lipid peroxidation and antioxidant-related indicators, decreased Fe~(2+) levels, alleviated fibrosis, and decreased the expression of TFR1, α-SMA, and collagen Ⅰ proteins in lung tissues, while increased the expression of GPX4, FTH1, Nrf2, and xCT proteins. In summary, ASR-AR ultrafiltration extract has an ameliorative effect on radiation-induced pulmonary fibrosis, and its mechanism may involve the inhibition of ferroptosis by regulating the Nrf2/xCT/GPX4 signaling pathway.

Research on the mechanism of shengxian and jinshuiliujun decoction in treating silicosis based on network pharmacology

Objective: To explore the active ingredients of shengxian and jinshuiliujun decoction with the method of network pharmacology, and to verify the experimental mechanism of its treatment of silicosis. Methods: In May 2023, the active ingredients and targets of drugs in shengxian and jinshuiliujun decoction were obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. The target of silicosis disease was screened by databases such as Genecards, Disease Gene Network (DisGeNET), Comparative Toxicogenomics Database (CTD), etc. The screened drug targets and disease targets were intersected to obtain the target set of shengxian and jinshuiliujun decoction for the treatment of silicosis. Protein-protein interaction (PPI) network analysis was performed on the target set through STRING database, and core target genes were screened. GO enrichment analysis and KEGG pathway analysis of intersection genes were performed based on Metascape database, and molecular docking verification of key components and targets of shengxian and jinshuiliujun decoction was carried out. Twenty-four adult male SD rats with SPF grade were randomly divided into control group, model group and TCM intervention group, with 8 rats in each group. The dust-stained rat model was prepared by non-tracheal exposure of 1 ml silica suspension (50 mg/ml) in one go, and TCM intervention group was given shengxian and jinshuiliujun decoction[6 g/ (kg·d) ] on the second day. The CT of the lungs of each group was observed 28 days after the dust-stained rat model. Paraffin sections of rat lung tissues were prepared and stained with Hematoxylin-Eosin (HE) and Masson. Western blot was used to verify the expression of core target-related proteins in rat lung tissues after the intervention of shengxian and jinshuiliujun decoction for 28 days, and the differences in protein expression between groups were compared by one-way analysis of variance. Results: A total of 205 active ingredients and 3345 active compounds were selected from shengxian and jinshuiliujun decoction, corresponding to 281 targets, among which 240 targets were related to silicosis. Serine/threonine kinase 1 (AKT1), tumor protein p53 (TP53), tumor necrosis factor (TNF) and interleukin (IL) 6 may be the key targets of shengxian and jinshuiliujun decoction in the treatment of silicosis. Through enrichment analysis, 30 GO entries and 20 potential signaling pathways were screened according to P-value, including nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) and cancer signaling pathways. Molecular docking showed that the active compounds of shengxian and jinshuiliujun decoction had good binding with the core target proteins, and the strongest binding properties were beta-sitosterol and TNF-α (-10.45 kcal/mol). In animal experiments, the inflammatory infiltration and fibrosis of lung tissue of rats in TCM intervention group were significantly improved. Compared with control group, the levels of TNF-α, IL-1β, IL-6 and NF-κB in lung tissue of model group were significantly increased (P<0.05). Compared with model group, the lung injury of rats in TCM intervention group was significantly improved, and the expressions of TNF-α, IL-1β, IL-6 and NF-κB were significantly decreased (P<0.05) . Conclusion: Shengxian and jinshuiliujun decoction in the treatment of silicosis may play an anti-fibrosis role by inhibiting the NF-κB signal transduction pathway mediated by inflammatory factors such as TNF-α and IL-1β, which provides a reference for further exploring the material basis and mechanism of its action.

Inhalation of hydrogen gas protects against mitomycin-induced pulmonary veno-occlusive disease

BackgroundAs a subtype of pulmonary hypertension (PH), pulmonary veno-occlusive disease (PVOD) is devastating and life-threatening disease without effective therapy. Hydrogen has been reported to exhibits antioxidant and anti-inflammatory effects in a rat model induced by monocrotaline of PH. In this study, we investigated the effects of inhaled hydrogen gas on the prevention and treatment of PVOD induced by mitomycin C (MMC) in rats.MethodsPVOD was induced in female Sprague-Dawley rats through intraperitoneal injection of MMC at a concentration of 3 mg·kg− 1·wk− 1 for 2 weeks. Inhalation of hydrogen gas (H2) was administered through a designed rat cage concurrently or two weeks after MMC administration. The severity of PVOD was assessed by using hemodynamic measurements and histological analysis. The expression levels of general control nonderepressible 2 (GCN2), nuclear factor erythroid 2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1) and endothelial-to-mesenchymal transition (EndoMT) related proteins in lung tissue were measured. Levels of lipid peroxidation pro-inflammatory cytokines in serum were determined.ResultsInhaled H2 improved hemodynamics and right heart function, reversed right ventricular hypertrophy, and prevented pulmonary vessel reconstitution in both prevention and treatment approaches. It decreased malondialdehyde (MDA) levels in the serum and the expression of NADPH oxidase 1 (NOX-1) in lung tissue. It regulated Nrf2/HO-1 signaling pathway and anti-inflammatory factor GCN2 in lung tissue, accompanied by a decrease in macrophages and pro-inflammatory cytokines. Our data suggested that H2 inhalation effectively countered EndoMT induced by MMC, as evidenced by the detection of endothelial markers (e.g., VE-cadherin and CD31) and mesenchymal markers (e.g., vimentin and fibronectin). Further research revealed that H2 preserved p-Smad3 and induced p-Smad1/5/9.ConclusionInhalation of H2 effectively inhibits the pathogenesis of PVOD induced by MMC in rats. This inhibitory effect may be attributed to the antioxidant and anti-inflammatory properties of H2.

Integration of microbiomics, metabolomics, and transcriptomics reveals the therapeutic mechanism underlying Fuzheng-Qushi decoction for the treatment of lipopolysaccharide-induced lung injury in mice

Ethnopharmacological relevanceFuzheng-Qushi decoction (FZQS) is a practical Chinese herbal formula for relieving cough and fever. Therefore, the action and specific molecular mechanism of FZQS in the treatment of lung injury with cough and fever as the main symptoms need to be further investigated. Aims of the studyTo elucidate the protective effects of FZQS against lung injury in mice and reveal its potential targets and key biological pathways for the treatment of lung injury based on transcriptomics, microbiomics, and untargeted metabolomics analyses. Materials and methodsLipopolysaccharide (LPS) was used to induce a mouse model of lung injury, followed by the administration of FZQS. ELISA was used to detect IL-1β, IL-6, IL-17A, IL-4, IL-10, and TNF-α, in mouse lung tissues. Macrophage polarization and neutrophil activation were measured by flow cytometry. RNA sequencing (RNA-seq) was applied to screen for differentially expressed genes (DEGs) in lung tissues. RT-qPCR and Western blot assays were utilized to validate key DEGs and target proteins in lung tissues. 16S rRNA sequencing was employed to characterize the gut microbiota of mice. Metabolites in the gut were analyzed using untargeted metabolomics. ResultsFZQS treatment significantly ameliorated lung histopathological damage, decreased pro-inflammatory cytokine levels, and increased anti-inflammatory cytokine levels. M1 macrophage levels in the peripheral blood decreased, M2 macrophage levels increased, and activated neutrophils were inhibited in mice with LPS-induced lung injury. Importantly, transcriptomic analysis showed that FZQS downregulated macrophage and neutrophil activation and migration and adhesion pathways by reversing 51 DEGs, which was further confirmed by RT-qPCR and Western blot analysis. In addition, FZQS modulated the dysbiosis of the gut microbiota by reversing the abundance of Corynebacterium, Facklamia, Staphylococcus, Paenalcaligenes, Lachnoclostridium, norank_f_Muribaculaceae, and unclassified_f_Lachnospiraceae. Meanwhile, metabolomics analysis revealed that FZQS significantly regulated tryptophan metabolism by reducing the levels of 3-Indoleacetonitrile and 5-Hydroxykynurenine. ConclusionFZQS effectively ameliorated LPS-induced lung injury by inhibiting the activation, migration, and adhesion of macrophages and neutrophils and modulating gut microbiota and its metabolites.

Role and mechanism of epithelial-mesenchymal transition in a rat model of bronchopulmonary dysplasia induced by hyperoxia exposure

To investigate the role and mechanism of epithelial-mesenchymal transition (EMT) in a rat model of bronchopulmonary dysplasia (BPD). The experiment consisted of two parts. (1) Forty-eight preterm rats were randomly divided into a normoxia group and a hyperoxia group, with 24 rats in each group. The hyperoxia group was exposed to 85% oxygen to establish a BPD model, while the normoxia group was kept in room air at normal pressure. Lung tissue samples were collected on days 1, 4, 7, and 14 of the experiment. (2) Rat type II alveolar epithelial cells (RLE-6TN) were randomly divided into a normoxia group (cultured in air) and a hyperoxia group (cultured in 95% oxygen), and cell samples were collected 12, 24, and 48 hours after hyperoxia exposure. Hematoxylin-eosin staining was used to observe alveolarization in preterm rat lungs, and immunofluorescence was used to detect the co-localization of surfactant protein C (SPC) and α-smooth muscle actin (α-SMA) in preterm rat lung tissue and RLE-6TN cells. Quantitative real-time polymerase chain reaction and protein immunoblotting were used to detect the expression levels of EMT-related mRNA and proteins in preterm rat lung tissue and RLE-6TN cells. (1) Compared with the normoxia group, the hyperoxia group showed blocked alveolarization and simplified alveolar structure after 7 days of hyperoxia exposure. Co-localization of SPC and α-SMA was observed in lung tissue, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 7 and 14 days of hyperoxia exposure compared to the normoxia group. In the hyperoxia group, the mRNA and protein levels of TGF-β1, α-SMA, and N-cadherin were increased, while the mRNA and protein levels of SPC and E-cadherin were decreased at 7 and 14 days of hyperoxia exposure compared to the normoxia group (P<0.05). (2) SPC and α-SMA was observed in RLE-6TN cells, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 24 and 48 hours of hyperoxia exposure compared to the normoxia group. Compared to the normoxia group, the mRNA and protein levels of SPC and E-cadherin in the hyperoxia group were decreased, while the mRNA and protein levels of TGF-β1, α-SMA, and E-cadherin in the hyperoxia group increased at 48 hours of hyperoxia exposure (P<0.05). EMT disrupts the tight connections between alveolar epithelial cells in a preterm rat model of BPD, leading to simplified alveolar structure and abnormal development, and is involved in the development of BPD. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 765-773.

CCDC59 alleviates bleomycin-induced inflammation and pulmonary fibrosis by increasing SP-B and SP-C expression in mice

BackgroundPulmonary fibrosis is a progressive disease with high incidence and poor prognosis. It is urgent to explore new therapeutic methods for pulmonary fibrosis. As a new treatment method, gene therapy has attracted more and more attention. CCDC59 is a transcriptional coactivator of SP-B and SP-C. Our study mainly aims to explore the effect of overexpression of CCDC59 gene in pulmonary fibrosis of mice. MethodsCCDC59 overexpressing lentivirus was constructed and then concentrated. RT-qPCR, Western blotting, and immunofluorescence assays were used to detect the expression of CCDC59, SP-B and SP-C protein in cell line and lung tissues after infected with lentivirus. Immunohistochemical staining and hematoxylin-eosin staining assays were used to assess the degree of fibrosis and ELISA assay was used to detect the concentrations of inflammatory factors, SP-B, and SP-C in bronchoalveolar lavage fluid of mice. Dynamic changes of mice lung function at various time points were assessed by lung function test assay. HIPPO pathway and proliferation capacity of alveolar type II epithelial cells were evaluated by immunofluorescence staining and Western blotting. ResultsResults showed that endotracheal instillation of CCDC59 overexpressed lentivirus significantly alleviated bleomycin-induced inflammation and pulmonary fibrosis in mice. Overexpression of CCDC59 protein in type II alveolar epithelial cells can enhance the expression of SP-B and SP-C. Overexpression of CCDC59 protein significantly protected against pulmonary inflammatory response and improved lung function of mice. Overexpression of CCDC59 protein significantly alleviated the hyperactivation of HIPPO pathway and increased the proliferative capacity of type II alveolar epithelial cells in lung. ConclusionCCDC59 can alleviate inflammation and pulmonary fibrosis in mice by upregulating the expression of SP-B and SP-C in type II alveolar epithelial cells and alleviating the hyperactivation of HIPPO pathway. Our study offers a new potential treatment for pulmonary fibrosis.

Anti-inflammatory mixture alleviates acute lung injury induced by sepsis in rats by modulating Beclin-1-mediated autophagy

To investigate the protective effects of an anti-inflammatory mixture on acute lung injury (ALI) induced by sepsis in rats, as well as its possible mechanisms. A total of 40 Sprague-Dawley (SD) rats were randomly divided into the sham group, septic ALI model group (model group), 3-methyladenine (3-MA) control group, and anti-inflammatory mixture pretreatment group, with 10 rats in each group. Cecal ligation and perforation (CLP) was performed to reproduce a septic ALI model. The rats in the sham group only underwent opening and closing the abdomen without perforation and ligation. Both groups were given saline gavage and intraperitoneal injection for 3 consecutive days before surgery. The 3-MA control group was given intraperitoneal injection of saline and autophagy inhibitor 3-MA 15 mg/kg for 3 consecutive days before modeling. The anti-inflammatory mixture pretreatment group was given 8.8 mL/kg of anti-inflammatory mixture by gavage [the composition of anti-inflammatory mixture: rhubarb 15 g (after the next), coptis chinensis 15 g, baical skullcap root 12 g, magnoliae cortex 12 g, dahurian patrinia herb 30 g] and saline intraperitoneal injection for 3 consecutive days before modeling. The rats in each group were anesthetized 24 hours after surgery and died due to abdominal aortic blood collection. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum inflammatory cytokines interleukins (IL-1β and IL-6). Lung tissue was taken and then the bronchoalveolar lavage fluid (BALF) was collected, and the levels of IL-1β and IL-6 were detected by ELISA. Lung wet/dry weight (W/D) ratio was measured. After hematoxylin-eosin (HE) staining, the histopathological changes of the lungs were observed under light microscopy. Western blotting was used to detect the expression of autophagy markers microtubule-associated protein 1 light chain 3- II/I (LC3- II/I) and Beclin-1 protein in lung tissue. Autophagosomes in lung tissue were observed with transmission electron microscopy. Compared with the sham group, the rats in the model group exhibited severe destruction of lung tissue structure, with significant infiltration of inflammatory cells, the lung W/D ratio and the levels of IL-1β and IL-6 in serum and BALF were significantly increased, the expressions of LC3- II/I and Beclin-1 protein were down-regulated, the autophagosomes were more. The rats in the 3-MA control group exhibited more severe lung tissue injury as compared with the model group, the lung W/D ratio and the levels of inflammatory cytokines in serum and BALF were further increased, the expressions of LC3- II/I and Beclin-1 protein still showed a decrease tendency as compared with the sham group, and the autophagosomes were less than that in the model group. Compared with the model group, the anti-inflammatory mixture pretreatment group showed milder lung tissue injury with a minimal amount of inflammatory cell infiltration, the lung W/D ratio was significantly reduced (7.07±1.02 vs. 11.33±1.85, P < 0.05), the levels of IL-1β and IL-6 in both serum and BALF were significantly decreased [IL-1β (ng/L): 26.04±3.86 vs. 40.83±5.46 in serum, 17.75±2.02 vs. 26.86±4.32 in BALF; IL-6 (ng/L): 91.28±10.15 vs. 129.44±13.05 in serum, 76.06±7.51 vs. 120.91±7.47 in BALF, all P < 0.05], and the ratio of LC3- II/I and Beclin-1 protein expression were significantly increased [LC3- II/I ratio: 1.23±0.02 vs. 0.60±0.02, Beclin-1 protein (Beclin-1/GAPDH): 2.37±0.33 vs. 0.62±0.05, both P < 0.05]. Furthermore, an increase in the number of autophagosomes was observed. The anti-inflammatory mixture improves lung injury in rats with sepsis induced by CLP and reduce inflammation levels, potentially through upregulation of Beclin-1-mediated autophagy.

Effect of Peitu Yifei Granules on autophagy mechanism in rats with idiopathic pulmonary fibrosis based on PI3K/Akt/mTOR signaling pathway

To investigate the mechanism by which Peitu Yifei Granules inhibit idiopathic pulmonary fibrosis(IPF) in rats, fifty specific-pathogen-free(SPF) grade male Wistar rats were randomly divided into blank group and modeling group. IPF was induced in the modeling group rats by tracheal infusion of 5 mg·kg~(-1) bleomycin(BLM) and then randomly divided into model group, pirfenidone group, and high-dose, medium-dose, and low-dose groups treated with Peitu Yifei Granules. After 24 hours of modeling, the treatment groups received intragastric administration of either Peitu Yifei Granules or pirfenidone as a positive control drug; meanwhile, the model group received an equal volume of normal saline. After 21 days of treatment administration, lung tissue samples were collected for analysis. Pathological changes in lung tissues were assessed using hematoxylin-eosin(HE) staining and Masson's trichrome staining. The expression levels of protein kinase B(Akt), mammalian target of rapamycin(mTOR), their phosphorylated forms, and sequestosome 1(p62) were determined through Western blot(WB). Fluorescent quantitative real-time polymerase chain reaction(RT-qPCR) was used to measure messenger ribonucleic acid(mRNA) expression levels of Beclin-1, microtubule-associated proteins 1A/1B light chain 3B(LC3B), and p62. Immunohistochemistry was performed to assess protein expression levels of Beclin-1 and LC3B in lung tissue samples. RESULTS:: demonstrated that lung tissue structure appeared normal without significant collagen deposition in the blank group rats. In contrast, rats from the model group exhibited thickened alveolar septa along with evident inflammatory changes and collagen deposition. Compared to the model group rats, those treated with Peitu Yifei Granules or pirfenidone showed significantly improved lung tissue structure with reduced inflammation and collagen deposition observed histologically. Furthermore, compared with those of the blank group, the expressions of p62 and its mRNA, p-Akt and p-mTOR protein in lung tissues of the model group were significantly increased, while Beclin-1, LC3B and their mRNA levels were significantly decreased. Compared with those of the model group, the expressions of p62 and its mRNA, p-Akt and p-mTOR in lung tissues of the pirfenidone group and Peitu Yifei Granules high-dose and medium-dose groups were significantly decreased, while Beclin-1, LC3B and their mRNA expressions were significantly increased. The above results indicate that Peitu Yifei Granules can improve autophagy levels in lung tissues by inhibiting the phosphoinositide 3-kinase(PI3K)/Akt/mTOR signaling pathway and delay the development of IPF disease.

Exploring the Mechanism of Zhishi-Xiebai-Guizhi Decoction for the Treatment of Hypoxic Pulmonary Hypertension Based on Network Pharmacology and Experimental Analyses.

Hypoxic Pulmonary Hypertension (HPH), a prevalent disease in highland areas, is a crucial factor in various complex highland diseases with high mortality rates. Zhishi-Xiebai-Guizhi Decoction (ZXGD), traditional Chinese medicine with a long history of use in treating heart and lung diseases, lacks a clear understanding of its pharmacological mechanism. This study aimed to investigate the pharmacological effects and mechanisms of ZXGD on HPH. We conducted a network pharmacological prediction analysis and molecular docking to predict the effects, which were verified through in vivo experiments. Network pharmacological analysis revealed 51 active compounds of ZXGD and 701 corresponding target genes. Additionally, there are 2,116 target genes for HPH, 311 drug-disease co-target genes, and 17 core target genes. GO functional annotation analysis revealed that the core target genes primarily participate in biological processes such as apoptosis and cellular response to hypoxia. Furthermore, KEGG pathway enrichment analysis demonstrated that the core targets are involved in several pathways, including the phosphatidylinositol- 3 kinase/protein kinase B (PI3K/Akt) signaling pathway and Hypoxia Inducible Factor 1 (HIF1) signaling pathway. In vivo experiments, the continuous administration of ZXGD demonstrated a significant improvement in pulmonary artery pressure, right heart function, pulmonary vascular remodeling, and pulmonary vascular fibrosis in HPH rats. Furthermore, ZXGD was found to inhibit the expression of PI3K, Akt, and HIF1α proteins in rat lung tissue. In summary, this study confirmed the beneficial effects and mechanism of ZXGD on HPH through a combination of network pharmacology and in vivo experiments. These findings provided a new insight for further research on HPH in the field of traditional Chinese medicine.

Schistosoma japonicum cystatin alleviates paraquat poisoning caused acute lung injury in mice through activating regulatory macrophages

BackgroundParaquat (PQ) is a widely used herbicide that poisons human by accident or intentional ingestion. PQ poisoning causes systemic inflammatory response syndrome (SIRS) resulting in acute lung injury (ALI) with an extremely high mortality rate. Blood trematode Schistosoma japonicum-produced cystatin (Sj-Cys) is a strong immunomodulatory protein that has been experimentally used to treat inflammation related diseases. In this study, Sj-Cys recombinant protein (rSj-Cys) was used to treat PQ-induced lung injury and the immunological mechanism underlying the therapeutic effect was investigated. MethodsPQ-induced acute lung injury mouse model was established by intraperitoneally injection of 20 mg/kg of paraquat. The poisoned mice were treated with rSj-Cys and the survival rate was observed up to 7 days compared with the group without treatment. The pathological changes of PQ-induced lung injury were observed by examining the histochemical sections of affected lung tissue and the wet to dry ratio of lung as a parameter for inflammation and edema. The levels of the inflammation related cytokines IL-6 and TNF-α and regulatory cytokines IL-10 and TGF-β were measured in sera and in affected lung tissue using ELISA and their mRNA levels in lung tissue using RT-PCR. The macrophages expressing iNOS were determined as M1 and those expressing Arg-1 as M2 macrophages. The effect of rSj-Cys on the transformation of inflammatory M1 to regulatory M2 macrophages was measured in affected lung tissue in vivo (EKISA and RT-PCR) and in MH-S cell line in vitro (flow cytometry). The expression levels of TLR2 and MyD88 in affected lung tissue were also measured to determine their role in the therapy of rSj-Cys on PQ-induced lung injury. ResultWe identified that treatment with rSj-Cys significantly improved the survival rate of mice with PQ-induced lung injury from 30 % (untreated) to 80 %, reduced the pathological damage of poisoning lung tissue, associated with significantly reduced levels of proinflammatory cytokines (IL-6 from 1490 to 590 pg/ml, TNF-α from 260 to 150 pg/ml) and increased regulatory cytokines (IL-10 from360 to 550 pg/ml, and TGF-β from 220 to 410 pg/ml) in both sera (proteins) and affected lung tissue (proteins and mRNAs). The polarization of macrophages from M1to M2 type was found to be involved in the therapeutic effect of rSj-Cys on the PQ-induced acute lung injury, possibly through inhibiting TLR2/MyD88 signaling pathway. ConclusionsOur study demonstrated the therapeutic effect of rSj-Cys on PQ poisoning caused acute lung injury by inducing M2 macrophage polarization through inhibiting TLR2/MyD88 signaling pathway. The finding in this study provides an alternative approach for the treatment of PQ poisoning and other inflammatory diseases.

Effects of iris xanthin on airway inflammation, airway remodeling, and the HMGB1/TLR4/NF-κB pathway in asthmatic young mice

To explore the effects of iris xanthin on airway inflammation, airway remodeling, and the high mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway in asthmatic young mice. Sixty male BALB/c young mice were randomly assigned into six groups: a blank group, a model group, a dexamethasone group, and low, medium, and high dose groups of iris xanthin, with ten mice per group. Asthma models were induced through intraperitoneal injections of a sensitizing agent [ovalbumin (OVA) 20 μg + aluminum hydroxide gel 2 mg], followed by 4% OVA aerosol inhalation. Lung function was measured using a pulmonary function tester to determine lung volume (LV), resting ventilation per minute (VE), and airway reactivity (Penh value). Hematoxylin-eosin (HE) staining was employed to examine and analyze airway remodeling. The contents of interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid were quantified using ELISA. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis were used to assess the expression of HMGB1/TLR4/NF-κB pathway-related mRNA and proteins in lung tissues. Compared to the model group, the dexamethasone and iris xanthin-treated groups (low, medium, and high doses) exhibited significant increases in LV and VE (P<0.05), with incremental dose-dependent increases observed in the iris xanthin groups. Additionally, Penh values, IL-1β, IL-6, TNF-α, and airway remodeling indicators, along with mRNA levels of HMGB1, TLR4, and NF-κB p65 and protein levels of HMGB1, TLR4, and p-NF-κB p65, were all reduced (P<0.05) in a dose-dependent manner. When compared to the dexamethasone group, the low and medium dose iris xanthin groups showed decreases in LV and VE (P<0.05), whereas Penh values, IL-1β, IL-6, TNF-α, and airway remodeling indicators, along with mRNA levels of HMGB1, TLR4, NF-κB p65 and protein levels of HMGB1, TLR4, and p-NF-κB p65, were increased (P<0.05). No significant differences were noted in these indices between the high dose iris xanthin group and the dexamethasone group (P>0.05). Iris xanthin can effectively alleviates airway inflammation and inhibits airway remodeling in asthmatic young mice, possibly through the suppression of the HMGB1/TLR4/NF-κB pathway.

Effect of maternal cigarette smoke exposure on COPD progression in offspring mice

ObjectiveTo investigate the impact of maternal smoking on chronic obstructive pulmonary disease (COPD) progression in offspring. MethodsUsing female C57BL/6 J mice, a maternal cigarette smoke exposure (CSE) model was established. Mice were exposed to cigarette smoke for 2 hours/day, 7 days/week, with a minimum 4-hour interval between exposures. Experimental groups included control (Con), pregnancy exposure (AS), pre-pregnancy exposure (SA), and pre-pregnancy + pregnancy exposure (SS). Lung function tests (Penh, PAU, TVb, EF50, Tr) were conducted on male offspring at 7 weeks. Histopathology, electron microscopy, and protein level changes were examined. ResultsLung function tests revealed significant impairments in Penh, PAU, TVb, EF50, and Tr in offspring across all exposure scenarios. Specifically, AS experienced significant lung function impairment and mitochondrial dysfunction in offspring, with noticeable pulmonary lesions and increased apoptosis. SA showed similar or even more severe lung function impairment and cellular apoptosis. SS exhibited the most pronounced effects, with the highest levels of lung dysfunction, mitochondrial damage, and apoptosis. Histopathological analysis showed pulmonary lesions in offspring exposed to maternal CSE. Flow cytometry revealed increased apoptosis and reduced mitochondrial membrane potential in offspring lung cells. Electron microscopy confirmed mitochondrial dysfunction. Upregulation of apoptotic proteins and downregulation of anti-apoptotic protein Bcl-2 were found in offspring lung tissue exposed to maternal CSE. ConclusionMaternal smoking induces impaired lung function, pulmonary lesions, and mitochondrial dysfunction in offspring, regardless of exposure timing and duration. Additionally, it alters expression of apoptosis-related proteins in offspring lung tissue, potentially contributing to COPD susceptibility.

Anthrahydroquinone‑2,6‑disulfonate attenuates PQ‑induced acute lung injury through decreasing pulmonary microvascular permeability via inhibition of the PI3K/AKT/eNOS pathway.

In paraquat (PQ)‑induced acute lung injury (ALI)/ acute respiratory distress syndrome, PQ disrupts endothelial cell function and vascular integrity, which leads to increased pulmonary leakage. Anthrahydroquinone‑2,6‑disulfonate (AH2QDS) is a reducing agent that attenuates the extent of renal injury and improves survival in PQ‑intoxicated Sprague‑Dawley (SD) rats. The present study aimed to explore the beneficial role of AH2QDS in PQ‑induced ALI and its related mechanisms. A PQ‑intoxicated ALI model was established using PQ gavage in SD rats. Human pulmonary microvascular endothelial cells (HPMECs) were challenged with PQ. Superoxide dismutase, malondialdehyde, reactive oxygen species and nitric oxide (NO) fluorescence were examined to detect the level of oxidative stress in HPMECs. The levels of TNF‑α, IL‑1β and IL‑6 were assessed using an ELISA. Transwell and Cell Counting Kit‑8 assays were performed to detect the migration and proliferation of the cells. The pathological changes in lung tissues and blood vessels were examined by haematoxylin and eosin staining. Evans blue staining was used to detect pulmonary microvascular permeability. Western blotting was performed to detect target protein levels. Immunofluorescence and immunohistochemical staining were used to detect the expression levels of target proteins in HPMECs and lung tissues. AH2QDS inhibited inflammatory responses in lung tissues and HPMECs, and promoted the proliferation and migration of HPMECs. In addition, AH2QDS reduced pulmonary microvascular permeability by upregulating the levels of vascular endothelial‑cadherin, zonula occludens‑1 and CD31, thereby attenuating pathological changes in the lungs in rats. Finally, these effects may be related to the suppression of the phosphatidylinositol‑3‑kinase (PI3K)/protein kinase B (AKT)/endothelial‑type NO synthase (eNOS) signalling pathway in endothelial cells. In conclusion, AH2QDS ameliorated PQ‑induced ALI by improving alveolar endothelial barrier disruption via modulation of the PI3K/AKT/eNOS signalling pathway, which may be an effective candidate for the treatment of PQ‑induced ALI.

Effects and mechanism of Qingke Pingchuan granules against influenza virus infection.

Qingke Pingchuan granules (QPGs), which contain Houttuynia cordata Thunb, Fritillaria cirrhosa, fired licorice, and fired bitter almonds, among other components, can clear heat and ventilate the lungs, relieving cough and asthma. Clinically, QPGs are mainly used to treat cough, asthma, fever and other discomforts caused by acute or chronic bronchitis. In this study, the antiviral activity of QPGs against respiratory syncytial virus (RSV), influenza A virus A/FM/1/47 (H1N1), oseltamivir-resistant H1N1, A/Beijing/32/92 (H3N2), Sendai virus, and human adenovirus type 3 in Hep-2 or MDCK cells was evaluated using the CCK-8 method, and the cytotoxicity of QPGs to these two cell lines was tested. The effect of QPGs on mice infected with influenza A virus A/FM/1/47 (H1N1) was evaluated by measuring body weight, survival time, and survival rate, as well as virus titers and lesions in the lungs and levels of inflammatory factors in serum. In addition, the expression of TLR-7-My88-NF-κB signaling pathway-related proteins in lung tissues was analyzed by Western blotting and qRT-PCR. The results showed that QPGs had a potent inhibitory effect on the six viruses tested in vitro. Interestingly, QPGs also displayed particularly pronounced antiviral activity against H1N1-OC, similar to that of oseltamivir, a well-known antiviral drug. QPGs effectively protected mice from infection by H1N1, as indicated by significantly increased body weights, survival times, and survival rates and reduced lung virus titers of inflammatory factors and lung tissue injury. The levels of TLR-7-MyD88-NF-κB-pathway-related proteins in the lung tissue of infected mice were found to be decreased after QPG treatment, thereby alleviating lung injury caused by excessive release of inflammatory factors. Taken together, these findings indicate that QPGs have satisfactory activity against influenza virus infection.

JAK2 inhibitors improve RA combined with pulmonary fibrosis in rats by downregulating SMAD3 phosphorylation.

JAK inhibitors are well known for the treatment of rheumatoid arthritis (RA), but whether they can be used to treat pulmonary fibrosis, a common extra-articular disease of RA, remains to be clarified. A jak2 inhibitor, CEP33779 (CEP), was administered to a rat model of RA-associated interstitial lung disease to observe the degree of improvement in both joint swelling and pulmonary fibrosis. HFL1 cells were stimulated with TGF-β1 to observe the expression of p-JAK2. Then, different concentrations of related gene inhibitors (JAK2, TGFβ-R1/2, and p-STAT3) or silencers (STAT3, JAK2) were administered to HFL1 cells, and the expression levels of related proteins were detected to explore the underlying mechanisms of action. CEP not only reduced the degree of joint swelling and inflammation in rats but also improved lung function, inhibited the pro-inflammatory factors IL-1β and IL-6, reduced lung inflammation and collagen deposition, and alleviated lung fibrosis. CEP decreased the expression levels of TGFβ-R2, p-SMAD, p-STAT3, and ECM proteins in rat lung tissues. TGF-β1 induced HFL1 cells to highly express p-JAK2, with the most pronounced expression at 48 h. The levels of p-STAT3, p-SMAD3, and ECM-related proteins were significantly reduced after inhibition of either JAK2 or STAT3. JAK2 inhibitors may be an important and novel immunotherapeutic drug that can improve RA symptoms while also delaying or blocking the development of associated pulmonary fibrotic disease. The mechanism may be related to the downregulation of p-STAT3 protein via inhibition of the JAK2/STAT signaling pathway, which affects the phosphorylation of SMAD3.

Exploring the anti-inflammatory ingredients and potential of golden buckwheat (Fagopyrum dibotrys) on the TLR4/NLRP3 pathway in acute lung injury.

Golden buckwheat, also called Fagopyrum dibotrys (D. Don) H. Hara, is a plant of the genus Buckwheat in the buckwheat family. The aim of this study was to screen the bioactive ingredients of golden buckwheat extract and investigate the protective effect on acute lung injury (ALI). The ethyl acetate extract (EAE) was identified as the active fraction in LPS-induced RAW264.7 cells, with gallic acid, proanthocyanidin B2, and epicatechin at 0.0563%, 0.3707%, and 0.3868%, respectively. At the same time, 20 compounds (mainly flavonoids and organic acids) were identified by UPLC-Q-Exactive Orbitrap-HRMS in EAE. Furthermore, the EAE reduced lung histopathology scores in mice with ALI, decreased the dry-to-wet weight ratio of lung tissue, and significantly inhibited the concentrations of IL-1β, TNFα, and IL-6 in bronchoalveolar lavage fluid (BALF). It also reduced the number of leukocytes, decreased the activity of MPO in lung tissue, and inhibited the levels of TLR4/NLRP3 pathway mRNA and protein in lung tissue. Our study indicated that golden buckwheat as a source of functional food prevents or treats associated lung diseases by modulating the activation of the TLR4/NLRP3 signaling pathway.