Bronchoalveolar Lavage Fluid Of Rats Research Articles

Comparative analysis of cytology of rat bronchoalveolar lavage fluid after single exposure to metal oxide nanoparticles

Introduction. Human production activities (metallurgical, mining, electronics production and processing, batteries) are related to air pollution of the working area and the environments of complex composition aerosols. Among the aerosol components, ultrathin particles of the nanometer range (including metal nano-oxides) are considered to be the most dangerous. Due to their prevalence, study to assess the cytotoxicity of metal oxide nanoparticles are relevant.
 Material and methods. CuO, PbO, CdO, Fe2O3, NiO nanoparticle (NP) suspension were obtained by laser ablation. The study was done on white outbred female rats. A single intratracheal instillation of different chemical NPs was performed (in dose 0.5 mg/animal); control animals received a similar amount of deionized water. A day after the NP instillation bronchoalveolar lavage (BAL) was carried out with the subsequent assessment of its cytological indices.
 Results. The cytotoxic action of the studied NPs, based on the cytological indices of the BAL fluid, is changed as follows (from greater to lesser): CuO NP > CdO NP > PbO NP > NiO NP > Fe2O3 NP.
 Limitations. Such physical characteristics of nanoparticles as solubility in water and biological fluids, charge, adsorption capacity, resistance to aggregation, hydrophobicity, adhesion to surfaces, and the ability to generate free radicals have not been studied. Extrapolation of data from rodents to humans shall be done with caution, since cytotoxicity has been characterized only based on the main cellular parameters.
 Conclusion. Bronchoalveolar lavage cytology can be used as an effective screening method for the cytotoxic effect of NPs.

Small extracellular vesicles from irradiated lung epithelial cells promote the activation of fibroblasts in pulmonary fibrosis

Background Alveolar epithelial injury and dysfunction are the risk factors for radiation-induced pulmonary fibrosis (RIPF). However, it is not clear about the relationship between RIPF and the small extracellular vesicles (sEV) secreted by irradiated alveolar epithelial cells. Based on the activation of fibroblasts, this study explored the role of sEV derived from alveolar epithelial cells in RIPF and the potential mechanisms. Methods Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were used to characterize sEV. Western blotting was used to detect fibrosis-associated proteins. Cell counts and transwell assays were used to evaluate the proliferation and migration ability of fibroblasts. RT-PCR was used to observe the extracellular matrix (ECM) synthesized by fibroblasts, miRNA changes in the sEV were determined by second-generation sequencing. Results TEM, NTA, and western blotting showed the extracellular vesicles with a double-layer membrane structure of approximately 100 nm in diameter. The sEV derived from irradiated A549, HBEC3-KT, and MLE12 cells upregulated FN1 and alpha-SMA proteins expression in fibroblasts and drove the fibroblast to myofibroblast transition, and the sEV from irradiated mouse bronchoalveolar lavage fluid (BALF) affirmed the same results. In addition, the sEV derived from irradiated alveolar epithelial cells significantly increased the migration ability of fibroblasts and the expression of extracellular matrix proteins such as FN1. The results of miRNA sequencing of sEV in BALF of rats with RIPF showed that the metabolic pathway may be important for miRNA to regulate the activation of fibroblasts. Conclusion The sEV derived from radiated pulmonary epithelial cells promote the activation, migration and extracellular matrix proteins expression of lung fibroblasts; miRNA in sEV may be an important molecular that affects the activation of lung fibroblasts.

Ophiopogonin A attenuates acute lung inflammation in Klebsiella pneumonia mice by regulating RELA (v-rel reticuloendotheliosis viral oncogene homolog A (avian))

Ophiopogonin are natural products from the roots of Ophiopogon japonicas with various pharmacological activities. This study explored the mechanism of ophiopogonin A to alleviate the acute inflammation of the lungs of Klebsiella pneumonia. 64 rats were equally assigned into blank control group, injury model group (intratracheal injection of Klebsiella pneumonia to establish Klebsiella pneumonia model), Ophiopogonin A group and Ophiopogonin A+RELA group (combined group, on the basis of injury model group, received RELA mediated ophiopogonin A), with the dose of 10 mg/kg. The histopathological damage was observed under the microscope after HE staining, as well as inflammatory cells in bronchoalveolar lavage fluid (BALF), cytokine levels, and expression of RELA. After HE staining, compared with injury model group, bronchial epithelium structure in Ophiopogonin A group and combination group were relatively intact, the degree of infiltration of inflammatory cells was reduced, and the thickness of alveolar septum was reduced, especially in combination group. The pathological scores of Ophiopogonin A group and combination group were significantly lower, with a further lower score in combination group. The number of inflammatory cells in BALF of rats in Ophiopogonin A group and combination group were significantly reduced, especially in combination group. Similarly, cytokine levels in BALF supernatant and serum of rats in treatment group were reduced, especially in combination group (P < 0.05). Ophiopogonin A could down-regulate RELA, which was further reduced in combination group. In conclusion, ophiopogonin A can reduce the invasion and pathological damage of pathogens to lung tissue and exert an anti-inflammatory ability.

Features of the cytogram and cytokine profile of bronchoalveolar lavage fluid in experimental metabolic syndrome

The aim of the study was to identify the features of the cellular composition and cytokine profile of bronchoalveolar lavage fluid in rats in a model of diet-induced metabolic syndrome.Materials and methods. In an experiment on animals (rats), a model of metabolic syndrome (MS) induced by a high-fat and high-carbohydrate diet was reproduced. To assess the viability of the reproduced model, biochemical and morphometric methods were used, such as measurement of body weight, specific gravity of liver and visceral fat, and blood pressure, determination of glucose concentration in the blood (including a glucose tolerance test), as well as determination of blood lipid parameters. To assess the intensity of the inflammatory response in the blood, the concentration of total protein, the total number of leukocytes, and the levels of immunocytokines (interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)α, monocyte chemoattractant protein (MCP)-1) were determined. Open bronchoalveolar lavage was performed on the isolated heart – lung complex. The concentration of protein, immunocytokines (IL-6, IL-10, TNFα, MCP-1), the total number of leukocytes, and the ratio of their morphological types were determined in the bronchoalveolar lavage fluid (BALF).Results. In animals with MS, an increase in the total number of leukocytes in the blood due to granulocytes and a rise in the concentration of protein, TNFα, and IL-10 were revealed compared with the parameters in the controls. BALF analysis revealed an increase in the concentration of protein, the total number of leukocytes, and the absolute number of alveolar macrophages, neutrophil granulocytes, and lymphocytes. The levels of IL-6 and MCP-1 were more than 1.5 times higher.Conclusion. Changes in the qualitative and quantitative parameters of BALF are inflammatory in nature and are formed during a systemic inflammatory response accompanying metabolic disorders in modeling MS in rats in the experiment.

Allergen-specific immunotherapy with Alutard SQ improves allergic inflammation in house-dust mites-induced allergic asthma rats through inactivation of the HMGB1/TLR4/NF-κB pathway.

Allergen-specific immunotherapy (AIT) is the only available safe, effective, and long-term treatment for allergic airway diseases, including allergic asthma. However, the potential molecular mechanism of AIT in ameliorating airway inflammation remains unknown. Rats were sensitized and challenged with house dust mite (HDM) and administered with Alutard SQ or/and high mobility group box 1 (HMGB1) inhibitor, ammonium glycyrrhizinate (AMGZ) or HMGB1 lentivirus. The total and differential cell counts in rat bronchoalveolar lavage fluid (BALF) were detected. Hematoxylin and eosin staining (H&E) was performed to examine the pathological lesions in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the expression of inflammatory factors in lungs, BALF, and serum. Quantitative real-time PCR (qRT-PCR) was used to measure the levels of inflammatory factors in the lungs. Western blot assay was used to evaluate the expression of HMGB1, Τoll-like receptor 4 (TLR4), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the lungs. Consequently, AIT with Alutard SQ attenuated airway inflammation, the total and differential cells in BALF, and expression of Th (T helper)2 related cytokines and transforming growth factor beta 1 (TGF-β1). The regimen also upregulated Th-1-related cytokine expression by inhibiting the HMGB1/TLR4/NF-κB pathway in HDM-induced asthmatic rats. Furthermore, AMGZ, a HMGB1 antagonist, amplified the functions of AIT with Alutard SQ in the asthma rat model. Nevertheless, overexpression of HMGB1 reversed the functions of AIT with Alutard SQ in the asthma rat model. In summary, this work demonstrates the role of AIT with Alutard SQ, which inhibits the HMGB1/TLR4/NF-κB signaling pathway in allergic asthma management.

Exosomal miRNAs contribute to coal dust particle-induced pulmonary fibrosis in rats

Coal workers’ pneumoconiosis (CWP) is a fatal occupational disease caused by inhalation of coal dust particles, which leads to progressive pulmonary fibrosis. Recently, as new signal carriers for intercellular communication, exosomal miRNAs have been validated in the pathogenesis of multiple diseases. However, the research on exosomal miRNAs in CWP is still in the preliminary stage. Here, using miRNA sequencing, exosomal miRNA profiles in bronchoalveolar lavage fluid (BALF) from rats with pulmonary fibrosis induced by coal dust particles were analyzed, and the underlying biological function of putative target genes was explored by GO term analysis and KEGG pathway enrichment analysis. According to the results, intratracheal instillation of coal dust particles can alter the exosomal miRNAs expression in the BALF of rats. Further bioinformatics analysis provided some clues to reveal their function in pathological process of pneumoconiosis. More importantly, we identified 4 differentially expressed exosomal miRNAs (miRNA-21–5p, miRNA-29a-3p, miRNA-26a-5p, and miRNA-34a-5p) by qRT‑PCR and further verified the temporal changes in the expression of these exosomal miRNAs in animal models from 2 weeks to 16 weeks postexposure. In addition, we conducted a preliminary study on Smad7 as a potential target of miRNA-21–5p and found that exosomal miRNA 21–5p/Smad7 may contribute to the pulmonary fibrosis induced by coal dust particles. Our study confirmed the contribution of exosomal miRNAs to coal dust particle-induced pulmonary fibrosis and provided new insights into the pathogenesis of CWP.

Heparin-Binding Protein Aggravates Acute Lung Injury in Septic Rats by Promoting Macrophage M1 Polarization and NF-κB Signaling Pathway Activation.

Objective Heparin-binding protein (HBP) plays an important role in sepsis and is a prognostic biomarker in patients with sepsis, but the role of HBP in the pathogenesis of sepsis-associated acute lung injury (ALI) remains unclear. This study aimed to investigate the role of HBP in sepsis-induced ALI and its underlying molecular mechanisms. Methods The cecal ligation and puncture (CLP) model was used to induce ALI in mice and randomly divided into 4 groups: control group, CLP (rats treated with cecal ligation and puncture), HBP (rats treated with CLP and HBP injection), and HBP + UFH (rats treated with CLP and injection of HBP and unfractionated heparin). Subsequently, HBP expression in rat serum and lung tissues was detected by qRT-PCR, edema and pathological changes in lung tissue by lung wet-to-dry weight ratio (W/D) and HE staining, myeloperoxidase (MPO) and superoxide dismutase (SOD) activities in lung tissues by detection kits. Additionally, ELISA and western blot were applied for the determination of IL-6, TNF-α, and IL-1β expression in rat bronchoalveolar lavage fluid, and iNOS, Arg-1, Mrc1, NF-κBp65, IKKα, IκBα, and p-IκBα expression in lung tissues. Results The expression levels of HBP in serum and lung tissues of rats in the HBP group were significantly increased, the lung tissues were severely injured, accompanied by a significant increase in MPO activity but a significant decrease in SOD activity, and the levels of IL-6, TNF-α, and IL-1β in bronchoalveolar lavage fluid were significantly increased. In addition, the expression levels of iNOS, NF-κB p65, IKKα, and p-IκBα in the lung tissues of rats in the HBP group were significantly increased, while the addition of unfractionated heparin reversed the above results. Conclusion HBP aggravates ALI in septic rats, and its mechanism may be related to the promotion of macrophage M1 polarization and activation of the NF-κB signaling pathway.

Effects of membrane-type PBLs on the expression of TNF-α and IL-2 in pulmonary tissue of SD rats after LPS-induced acute lung injury.

As the first target organ, the lungs usually display symptoms of acute lung injury (ALI). Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-2, are crucial in triggering the systemic inflammatory response syndrome and the subsequent cascading effects. Therefore, the inhibition of the release of inflammatory mediators has become an important strategy for the prevention and treatment of ALI. To evaluate the preventive and therapeutic effects of transmembrane peripheral blood leukocytes (PBLs) on lipopolysaccharide (LPS)-induced ALI and its mechanism. Sixty Sprague Dawley rats were randomly divided into experimental and control groups. The animal model was established through intravenous injection of LPS. Plasmid PBLs were dissolved in a saline solution and injected into the experimental group of rats in different doses (0.1 mg, 0.2 mg and 0.3 mg per rat) using the in situ injection method. After injecting the PBL solution, the rats were killed after 12 h, 24 h, 36 h, or 48 h. The expression of microRNA (miRNA)-25 and miRNA-223 was detected using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Tumor necrosis factor alpha and IL-2 levels in bronchoalveolar lavage fluid (BALF) were detected with an enzyme-linked immunosorbent assay (ELISA). The expressions of TNF-α and IL-2 proteins in lung tissue were detected using western blotting. The expression of miRNA-25 was upregulated in tissues and BALF in a doseand time-dependent manner, while miRNA-223 was downregulated. The differences were statistically significant compared to the control group (p < 0.05). The TNF-α and IL-2 levels in the BALF of rats in the experimental group were increased in a dose-dependent manner compared to the control group (p < 0.05). In the presence of PBLs, the expressions of TNF-α, IL-2, miRNA, and proteins were inhibited. Thus, PBLs were found to alleviate pulmonary tissue damage. In summary, PBLs have a protective effect on rats with ALI through the downregulation of TNF-α and IL-2 expression.

Tetrahydropalmatine induces the polarization of M1 macrophages to M2 to relieve limb ischemia-reperfusion-induced lung injury via inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Tetrahydropalmatine (THP) is the main component of the Chinese medicine Corydalis yanhusuo, which has been reported to alleviate limb ischemia-reperfusion-induced acute lung injury (LIR-ALI). This study aimed to investigate the mechanism underlying the effect of THP on relieving LIR-ALI. LIR-ALI model was established in rats with the presence or absence of THP pretreatment. Then, BEAS-2B cells and THP-1 macrophages were cocultured with rat serum from the Sham group and the Model group in the presence or absence of THP pretreatment. Subsequently, lung/body weight and lung wet/dry ratio of rats were calculated. Histological changes of lung tissues were observed by hematoxylin-eosin staining. Expression of CD86 and CD163 in lung tissues of rats was assessed by quantitative reverse transcription polymerase chain reaction, immunohistochemistry staining, and flow cytometry analysis. Levels of inflammatory cytokines were measured by enzyme linked immunosorbent assay. The expression of proteins related to toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/NLRP3 signaling was detected by western blot analysis. Results revealed that THP significantly relieved LIR-ALI in rats. Moreover, THP also reduced CD86 expression but elevated CD163 expression in lung tissues of rats with LIR-ALI. Furthermore, THP inhibited inflammation in serum and bronchoalveolar lavage fluid of rats with LIR-ALI and inactivated the TLR4/NF-κB/NLRP3 signaling in vivo. Additionally, coculture of serum from rats in the Model group also reduced viability, promoted inflammation, inactivated TLR4/NF-κB/NLRP3 expression in BEAS-2B cells and inhibited macrophage polarization, while these effects were all reversed by THP treatment. Collectively, THP could induce the polarization of M1 macrophage to M2 to suppress inflammation via inhibiting TLR4/NF-κB/NLRP3 signaling, thereby attenuating LIR-ALI.

Effects of transplanted mesenchymal stem cells on repair of the lung tissue of rats with experimental pulmonary fibrosis

Pulmonary fibrosis is one of the commonest forms of interstitial lung diseases with poorly studied methods of its treatment in both human and veterinary medicines. Therefore, this paper focused on seeking alternative methods of its diagnostics and treatment. The article provides the results of the study of bronchoalveolar lavage fluid of rats with experimental lung fibrosis and influence of transplanted allogeneic mesenchymal stem cells of the bone marrow on stimulation of regenerative processes in damaged lung tissues. The studies were conducted on female Wistar rats with pulmonary fibrosis modeled using single transthoracic injection of solution of bleomycin hydrochloride. For the purpose of treatment, we used allogeneic mesenchymal stem cells introduced by various methods and the traditional treatment. We determined that best normalization of the parameters of the studied brochoalveolar lavage occurred in animals that received mesenchymal stem cells. The most active repair processes were in the experimental group that received the mesenchymal stem cells directly to the lung tissue. The animals that received intravenous injection of mesenchymal stemm cells were observed to have lower clinical parameters of the brochoalveolar lavage, but still better than such in the group treated traditionally. The lowest parameters were in animals that received the traditional treatment; they were greater than the phisological parameters, but significantly exceeded them in animals of the control group, indicating presence of inflammatory process in the lung tissue. The conducted cytological assays of the samples of the brochoalveolar lavage revealed that experimental animals with experimental pulmonary fibrosis had development of macrophage and lymphocytic reactions under the influence of transplanted mesenchymal stemm cells. We observed no atypical cells in all the experimental groups. This allows us to draw a conclusion that using stem cells by various methods of transplantation does not stimulate the onset of negative reactons (formation of atypical cells, metastatic processes, etc). Thus, the results of the study of the influence of transplanted mesenchymal stem cells demonstrate that in the conditions of experimental pulmonary fibrosis, the activity of regenerative processes in pathologically altered lung tissue may be an effective method of treatment of animals with this kind of pathology.

Salidroside attenuates sepsis-associated acute lung injury through PPP1R15A mediated endoplasmic reticulum stress inhibition

BackgroundThis study explored the role and mechanism of salidroside in sepsis-associated acute lung injury (ALI). MethodSepsis-associated ALI in rats was induced by cecal ligation and perforation method, while lipopolysaccharide (LPS) was used to stimulate cell model. After the treatment of salidroside and dexamethasone (DEX), hematoxylin and eosin staining was applied to evaluate the rat lung injury. Next, the number of total cells and neutrophils in rat bronchoalveolar lavage fluid (BALF) was counted, and the lung wet/dry (W/D) ratio and water content were measured. The neutrophil elastase activity and inflammatory factor levels in BALF were detected by Elastase Assay kit and ELISA. The expressions of PPP1R15A and endoplasmic reticulum (ER) stress-related genes were detected by quantitative reverse transcription polymerase chain reaction and western blot. And the effects of silenced PPP1R15A and tauroursodeoxycholic acid (TUDCA) on cell viability, apoptosis and endoplasmic reticulum (ER) stress were determined by cell counting kit-8 assay, flow cytometry and western blot. ResultThe lung injury, inflammation and edema in sepsis model rats were alleviated by salidroside and DEX, meanwhile salidroside increased the viability and inhibited apoptosis in LPS-treated cells. The expression of PPP1R15A was decreased in sepsis models and increased by salidroside, and salidroside down-regulated the ER stress-related protein expressions in vitro and in vivo. Silenced PPP1R15A reversed the effect of salidroside on cell viability, apoptosis and ER stress, whereas TUDCA could counteract the above effect of silenced PPP1R15A. ConclusionSalidroside targeted PPP1R15A to ameliorate lung injury in sepsis through inhibiting ER stress.

Effect of curcumin on lung epithelial injury and ferroptosis induced by cigarette smoke

Cigarette smoke (CS)-caused ferroptosis was involved in the pathogenesis of COPD, but the role of ferroptosis in lung epithelial injury and inflammation is not clear. Rats were treated with CS or CUR and BEAS-2B cells were exposed to CS extract (CSE), ferrostatin-1 (Fer-1), deferoxamine (DFO), or CUR to detect reactive oxygen species (ROS) accumulation, lipid peroxidation, iron overload, and ferroptosis-related protein, which were the characteristic changes of ferroptosis. Compared with the control group, CSE-treated BEAS-2B cells had more cell death, higher cytotoxicity, and lower cell viability. The infiltration of inflammatory cell around the bronchi in the CS group of rats was more than that in the normal group. Meanwhile, CSE/CS elevated the levels of interleukin-6 and tumor necrosis factor-α in BEAS-2B cells and bronchoalveolar lavage fluid of rats. Besides, accumulative ROS and depleted glutathione was observed in vitro. In BEAS-2B cells and lung tissues of rats, CSE/CS increased malondialdehyde and iron; down-regulated solute carrier family 7, glutathione peroxidase 4, and ferritin heavy chain levels; and up-regulated transferrin receptor level. These changes were rescued by pretreatment of Fer-1 or DFO in vitro, and mitigated by CUR in vitro and in vivo. Collectively, this study reveals that ferroptosis was involved in lung epithelial cell injury and inflammation induced by CS, and CUR may alleviate CS-induced injury, inflammation, and ferroptosis of lung epithelial cell.

MUC5B regulates the airway inflammation induced by atmospheric PM2.5 in rats and A549 cells

Atmospheric PM2.5 can induce airway inflammation and mucin secretion. MUC5B is required for airway defense. However, the research on the role of MUC5B in airway inflammation induced by atmospheric PM2.5 remains limited. This study was designed to explore the role of MUC5B in airway inflammation induced by atmospheric PM2.5. In vivo, Wistar rats were exposed to 0, 1.5, 7.5, 37.5 mg/ kg PM2.5 saline suspension via intratracheal instillation. HE staining and AB-PAS staining were used to observe the airway inflammation and goblet cell hyperplasia. In vitro, normal A549 cells and MUC5B-knockdown A549 cells were exposed to 0, 100, 200 and 400 μg/mL PM2.5 for 6 h, 12 h, 24 h and 48 h. ELISA was used to measure the levels of TNF-α and IL-1β in serum and bronchoalveolar lavage fluid of rats and in cell culture. Real time-PCR and ELISA were used to quantify the mRNA and protein levels of MUC5B in trachea and lung of rats and in A549 cells. PM2.5 could cause the infiltration of inflammatory cells and increase the mucus secretions and goblet cell metaplasia. MUC5B is related to rats’ airway inflammation induced by PM2.5. A549 cells exposed to PM2.5 in higher concentration and longer time, the protein level of MUC5B was significantly increased, while the levels of IL-1β, TNF-α and MUC5B mRNA were significantly decreased. Compared with normal A549 cells, the levels of IL-1β and TNF-α were significantly higher in Muc5b-knockdown cells. Atmospheric PM2.5 can induce airway inflammation and mucin secretion. MUC5B played a critical role in controlling the inflammatory response induced by PM2.5.

Mesenchymal stem cells ameliorate silica-induced pulmonary fibrosis by inhibition of inflammation and epithelial-mesenchymal transition.

Silicosis is a devastating occupational disease caused by long‐term inhalation of silica particles, inducing irreversible lung damage and affecting lung function, without effective treatment. Mesenchymal stem cells (MSCs) are a heterogeneous subset of adult stem cells that exhibit excellent self‐renewal capacity, multi‐lineage differentiation potential and immunomodulatory properties. The aim of this study was to explore the effect of bone marrow‐derived mesenchymal stem cells (BMSCs) in a silica‐induced rat model of pulmonary fibrosis. The rats were treated with BMSCs on days 14, 28 and 42 after perfusion with silica. Histological examination and hydroxyproline assays showed that BMSCs alleviated silica‐induced pulmonary fibrosis in rats. Results from ELISA and qRT‐PCR indicated that BMSCs inhibited the expression of inflammatory cytokines TNF‐α, IL‐1β and IL‐6 in lung tissues and bronchoalveolar lavage fluid of rats exposed to silica particles. We also performed qRT‐PCR, Western blot and immunohistochemistry to examine epithelial‐mesenchymal transition (EMT)–related indicators and demonstrated that BMSCs up‐regulate E‐cadherin and down‐regulate vimentin and extracellular matrix (ECM) components such as fibronectin and collagen Ⅰ. Additionally, BMSCs inhibited the silica‐induced increase in TGF‐β1, p‐Smad2 and p‐Smad3 and decrease in Smad7. These results suggested that BMSCs can inhibit inflammation and reverse EMT through the inhibition of the TGF‐β/Smad signalling pathway to exhibit an anti‐fibrotic effect in the rat silicosis model. Our study provides a new and meaningful perspective for silicosis treatment strategies.

SKP2-Promoted Ubiquitination of FOXO3 Promotes the Development of Asthma.

Asthma is a respiratory disease with a dramatically increasing incidence globally. The present study explored the roles of S-phase kinase-associated protein 2 (SKP2) and forkhead box O3 (FOXO3) in asthma and their involvement in the Krüppel-like factor 15-lipoprotein receptor-related protein 5 (KLF15-LRP5) axis. SKP2 expression in patients with asthma and OVA-induced asthmatic Sprague Dawley rats was detected by reverse transcription quantitative PCR and Western blot assays. Alterations in SKP2 and LRP5 expression were evaluated in OVA-induced asthmatic rats, followed by measurement of inflammatory cytokines using ELISA and airway resistance using a methacholine challenge test. We applied TGF-β1 to establish the airway smooth muscle cell (ASMC) proliferation model of asthma. The FOXO3 ubiquitination and changes in cell biological behaviors were detected using immunoprecipitation, MTT, and Annexin V/propidium iodide assays. Flow cytometry was adopted to detect cell cycle, and ELISA was used to measure the concentrations of IL-4, IL-5, IL-13, and IgE in rat bronchoalveolar lavage fluid. SKP2 was highly expressed and FOXO3 was poorly expressed in patients with asthma and in OVA-induced asthmatic rats. SKP2 silencing decreased IL-4, IL-5, IL-13, and IgE expression in rat bronchoalveolar lavage fluid, whereas SKP2 enhanced FOXO3 ubiquitination to upregulate KLF15, which bound to the LRP5 promoter in TGF-β1-induced ASMCs and increased LRP5 expression. SKP2 enhanced airway hyperresponsiveness and inflammation in the OVA-induced rat model and augmented TGF-β1-induced ASMC proliferation by inhibiting the FOXO3/KLF15/LRP5 axis. Additionally, overexpressed SKP2 resulted in reduced numbers of ASMCs in the G1 phase but increased numbers in the G2/M phase. Collectively, we show that SKP2 promotes FOXO3 ubiquitination to suppress the KLF15-LRP5 axis, thereby exacerbating asthma.

Dynamics in the content of the cytokines in the bronchoalveolar lavage fluid in rats after acute inhalation intoxication by clorine and pyrolysis products, containing hydrogen chloride

It is known that inhalation exposure to chlorine and hydrogen chloride leads to damage to the respiratory system up to the development of acute pulmonary edema in victims. No data on the mechanisms of development of pulmonary edema upon exposure to hydrogen chloride have been found in the available literature. The study was carried out on white outbred male rats, which were divided into 3 groups: Group I control; Group II animals were intoxicated with chlorine at a dose of 1.5 median lethal concentration (30 min); Group III animals were intoxicated with hydrogen chloride at a dose of 1.5 median lethal concentration (30 min). Immediately after exposure to the studied toxicants, as well as after 1, 3 and 6 h, the lung coefficient and the content of cytokines (interleukins-1, 6, 10 and interferon-) in the bronchoalveolar lavage fluid were determined in animals. It was revealed that an increase in the lung coefficient (p 0.05) in animals in groups II and III was accompanied by a significant increase (1.5 times) in the content of the studied cytokines in the bronchial-alveolar lavage fluid compared with animals in group I. III an increase (p 0.05) in the content of cytokines is recorded later only 3 hours after exposure, while it is significantly lower than in animals of group II at all studied periods. Thus, intoxication with hydrogen chloride leads to a slower development of pulmonary edema and an increase in the content of both pro (interleukins-1, 6) and anti-inflammatory cytokines (interleukin-10, interferon-) in the bronchial-alveolar lavage fluid compared to animals, exposed to chlorine intoxication.

Study of cytokines in peripheral blood and lung of rats exposed to hard metal dust

Objective: To study the dynamic changes of cytokines in bronchoalveolar lavage fluid (BALF) and serum of hard metal lung disease (HMLDR) rats. Methods: In March 2019, the rats were randomly divided into 6 groups, each group included 8 rats: control (C) group include 3 groups, hard metal (HM) group include 3 groups. 10 mg HM were administered in HM group by using the pulmonary endotracheal tube. After 4, 8 and 12 week, the BALF and serum were collected for the enzyme-linked immunosorbent assay (ELISA) of matrix metalloproteinase-1 (MMP-1) , tissue inhibitor of metalloproteinase-1 (TIMP-1) and tumor necrosis factor-alpha (TNF-α) . Results: There was no abnormality in behavior, diet and fur of rats in C and HM group at each exposure time. There was no significant difference in body weight between the two groups of rats (P>0.05) . Compared with the C group, the expression of MMP-1 in BALF of rats in HM group were significantly higher in all stages (4, 8 and 12 weeks after exposure) (P<0.05) , the expression of TIMP-1 in BALF of rats in HM group were significantly higher in 8 and 12 weeks after exposure (P<0.05) . However, there was no significant difference in serum MMP-1 and TIMP-1 levels between the two groups in each stage (P>0.05) . There was no significant difference in TNF-α. level in BALF and serum between C and HM group in all stages (P>0.05) . Conclusion: Expression of MMP-1 and TIMP-1 in BALF have reference value in the HMLD auxiliary diagnosis.

Yiqi Gubiao pill alleviates chronic obstructive pulmonary disease in rats via upregulation of klotho expression

Purpose: To investigate the effect of Yiqi Gubiao pill (YGP) on chronic obstructive pulmonary disease (COPD) in rats, and the mechanism of its action.Methods: Six groups of eight-week old male SPF-grade Wistar rats (n = 60) were used (10 rats/group), viz, control, COPD, low-dose YGP, medium-dose YGP, high-dose YGP, and dexamethasone groups. Chronic obstructive pulmonary disease (COPD) was induced using a standard method. Graded doses of YGP were prepared by dissolving 100, 200 or 400 pills of Yiqi Gubiao. Lung damage and airway remodeling were determined by histological examination. Enzyme-linked immunosorbent assay (ELISA) was used to measure levels of klotho in rat serum and bronchoalveolar lavage fluid (BALF). The mRNA and protein expressions of klotho in lung tissue were assayed with qRT-PCR) and Western blot assay, respectively.Results: Serum and BALF klotho levels were significantly lower in COPD rats than in control, but the levels were markedly and dose-dependently increased by YGP (p &lt; 0.05). Treatment of COPD rats with YGP led to significant and dose-dependent upregulation of mRNA and protein expressions of klotho (p &lt; 0.05). Moreover, histologic score (index of lung damage) increased in COPD rats, relative to control, but it was dose-dependently decreased by YGP (p &lt; 0.05).Conclusion: These results suggest that YGP alleviates COPD in rats via the upregulation of klotho expression. However, the potential use of YGP for COPD still needs further clinical research.&#x0D; Keywords: Chronic obstructive pulmonary disease, Klotho, Lungs, Protein expression, Yiqi Gubiao pill

Pulmonary fate and consequences of transferrin-functionalized gold nanoparticles.

Surface functionalization of nanoparticles (NPs) may alter their biological interactions such as uptake by alveolar macrophages (AMs). Pulmonary delivery of gold NPs (Au NPs) has theranostic potential due to their optoelectronic properties, minimal alveoli to blood translocation, and possibility of specific cell targeting. Here, we examined whether coating Au NPs with transferrin alters their protein corona, uptake by macrophages, and pulmonary translocation.Methods: Rats were intratracheally instilled with transferrin-coated Au NPs (Tf-Au NPs) or polyethylene glycol-coated Au NPs (PEG-Au NPs). AMs were collected and processed for quantitation of Au cell uptake using ICP-MS and electron microscopy. Au retention in the lungs and other organs was also determined. The uptake of fluorescently labeled Tf-Au NPs and PEG-Au NPs by monocyte-derived human macrophages was also evaluated in vitro.Results: We showed that Tf-Au NPs were endocytosed by AMs and were retained in the lungs to a greater extent than PEG-Au NPs. Both Au NPs acquired similar protein coronas after incubation in rat broncho-alveolar lavage fluid (BALf). The translocation of Au from both NPs to other organs was less than 0.5% of the instilled dose. Transferrin coating enhanced the uptake of Au NPs by primary monocyte-derived human macrophages.Conclusions: We report that coating of NP surface with transferrin can target them to rat AMs and human monocyte-derived macrophages. NP functionalization with transferrin may enhance NP-based therapeutic strategies for lung diseases.

Protective Effect of Colchicine on LPS-Induced Lung Injury in Rats via Inhibition of P-38, ERK1/2, and JNK Activation

Introduction: Acute lung injury (ALI), a commonly detected syndrome, is characterized by the accumulation of neutrophils and leucocytes, and inflammation of pulmonary tissues. Objective: The present study was designed to investigate the effect and underlying mechanism of colchicine on LPS-induced lung injury. Methods: The rats were divided randomly into 6 groups of 10 each: normal control, untreated, and 4 colchicine (5, 10, 15, and 20 mg/kg) treatment groups. ALI was induced in rats by the administration of 20 μg LPS intratracheally. Rats in the normal control and untreated groups were injected normal saline, while those in the treatment groups received 5, 10, 15, and 20 mg/kg doses of colchicine daily for 1 month. ELISA was used for determination of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α, superoxide dismutase (SOD), and leucocytes in the rat bronchoalveolar lavage fluid (BALF). The expression of P-38, JNK, and Erk-1/2 was analysed by Western blotting. Results: In LPS-administered TC-1 cells, the levels of IL-1β, IL-6, and TNF-α were markedly higher. Treatment with colchicine reduced the levels of IL-1β, IL-6, and TNF-α in LPS-administered TC-1 cells. Colchicine treatment caused a marked reduction in LPS-induced accumulation of inflammatory cells in the rat lungs. The LPS-induced aggregation of leucocytes and neutrophils in the rat BALF was also suppressed markedly on treatment with colchicine. Treatment of the lung injury in rats with colchicine caused a marked decrease in the level of IL-1β, IL-6, and TNF-α in BALF. The LPS-mediated suppression of SOD in the rat BALF was prevented by treatment with colchicine. Treatment of the rats with colchicine attenuated the LPS-induced activation of P-38, Erk1/2, and JNK in pulmonary tissues. Conclusion: In summary, colchicine treatment prevents LPS-induced lung damage in rats through targeting activation of P-38, ERK1/2, and JNK. Therefore, colchicine may be used for the development of treatment for ALI.