Citrus peel extract protects against diesel exhaust particle-induced chronic obstructive pulmonary disease-like lung lesions and oxidative stress.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and characterized by emphysema, small airway remodeling and mucus hypersecretion. Citrus peels have been widely used as food spices and in traditional Chinese medicine for chronic lung disease. Given that citrus peels are known for containing antioxidants and anti-inflammatory compounds, we hypothesize that citrus peel intake can suppress oxidative stress and inflammatory response to air pollution exposure, thereby alleviating COPD-like pathologies. This study aimed to investigate the efficacy of citrus peel extract, namely Guang Chenpi (GC), in preventing the development of COPD induced by diesel exhaust particles (DEPs) and its potential mechanism. DEP-induced COPD-like lung pathologies, inflammatory responses and oxidative stress with or without GC treatment were examined in vivo and in vitro. Our in vivo study showed that GC was effective in decreasing inflammatory cell counts and inflammatory mediator (IL-17A and TNF-α) concentrations in bronchoalveolar lavage fluid (BALF). Pretreatment with GC extract also significantly decreased oxidative stress in the serum and lung tissue of DEP-induced COPD rats. Furthermore, GC pretreatment effectively reduced goblet cell hyperplasia (PAS positive cells) and fibrosis of the small airways, decreased macrophage infiltration as well as carbon loading in the peripheral lungs, and facilitated the resolution of emphysema and small airway remodeling in DEP-induced COPD rats. An in vitro free radical scavenging assay revealed robust antioxidant potential of GC in scavenging DPPH free radicals. Moreover, GC demonstrated potent capacities in reducing ROS production and enhancing SOD activity in BEAS-2B cells stimulated by DEPs. GC treatment significantly attenuated the increased level of IL-8 and MUC5AC from DEP-treated BEAS-2B cells. Mechanistically, GC treatment upregulated the protein level of Nrf-2 and could function via MAPK/NF-κB signaling pathways by suppressing the phosphorylation of p38, JNK and p65. Citrus peel extract is effective in decreasing oxidative stress and inflammatory responses of the peripheral lungs to DEP exposure. These protective effects further contributed to the resolution of COPD-like pathologies.

HPLC-DAD analysis of Quercus leucotrichophora extract and appraisal of its antiasthmatic potential via modulation of aquaporins, inflammatory, and oxidative stress biomarkers in Albino mice.

Herbal drugs offer an alternative approach for the treatment of diseases like asthma due to low cost and comparatively less adverse effects in contrast to synthetic drugs. Leaves of Quercus leucotrichophora are traditionally used for the treatment of asthma. The study was aimed to assess the anti-asthmatic activity of Quercus leucotrichophora (QL) methanolic (QLME) and aqueous extracts (QLAE) in ovalbumin-(OVA) induced asthma and chemical characterization of QL extract by High Performance Liquid Chromatography-Diode array detector (HPLC-DAD). Animals were inoculated with OVA (i.p) on day 1 and 14 followed by intranasal challenge on 27th and 29th day. Both extracts of QL at 600, 300 and 150 mg/kg and dexamethasone (2 mg/kg) l were administered consecutively from days 15-26 via oral gavage. The QL extracts notably reduced (p < 0.0001-p < 0.05) total and differential leukocyte counts in blood and BALF and serum IgE levels in contrast to disease control. Both extracts and Dex substantially improved activities of superoxide dismutase, catalase, and GSH, while reduced malondialdehyde level in treated mice. Treatment with extracts and Dex caused significant (p < 0.0001-p < 0.05) downregulation of tumor necrosis factor-α, interleukin-4, - 5, - 13, - 6, - 1ß, and NF-κB whereas, increased expression of Aquaporin (AQP) 1 and AQP5 in contrast to disease control. It was inferenced from findings that both extract of QL exhibited notable antiasthmatic potential might be due to presence of Daidzein-glucuronic acid, 3-Hydroxyphloretin 6'-hexoside, Catechin, Quercetin, and Kaemferol.

Hylocereus undatus flower extract suppresses OVA-induced allergic asthma in BALb/c mice by reducing airway inflammation and modulating gut microbiota.

Asthma is a chronic allergic respiratory disease with limited treatment options. Emerging findings indicate an important interaction between the gut microbiota and the lungs, and that the development of asthma causes changes in the gut environment. Hylocereus undatus flower (HUF) is a traditional Chinese medicine used in the treatment of pulmonary and intestinal diseases. Our previous studies have demonstrated significant anti-asthmatic and anti-inflammatory activity, but the exact mechanism has not been elucidated. In the current study, we validated the potential therapeutic asthma properties of HUF in vivo using an ovalbumin-induced allergic asthma mouse model. We found that HUF treatment significantly reduced the key features of allergic asthma, including an elevated respiratory rate, inflammatory cell accumulation, airway inflammation, and the expression of pro-inflammatory molecules. Histological analysis of mouse lungs showed that HUF attenuated lung inflammatory cell infiltration. Periodic acid-Schiff staining confirmed the reduced mucus secretion in lung mucosa, and Masson's staining confirmed the reduced collagen deposition in the lungs after HUF treatment. Western blot and immunohistochemistry confirmed that HUF increased lung SIRT1 and reduced p38MAPK, NF-κBp65, and caspase-1 proteins. 16 S rDNA sequencing showed that HUF improved the endostasis of the disrupted gut microbiota composition in asthmatic mice. Surprisingly, an inflammatory response was found in the gut of asthmatic mice, along with alterations in inflammation-associated SIRT1 and caspase-1 proteins, and HUF was able to ameliorate these lesions. In conclusion, these findings suggest that HUF may be a new drug candidate for the treatment of allergic asthma.

Processed product (Pinelliae Rhizoma Praeparatum) of Pinellia ternata (Thunb.) Breit. Alleviates the allergic airway inflammation of cold phlegm via regulation of PKC/EGFR/MAPK/PI3K-AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Pinelliae Rhizoma Praeparatum (PRP) is a traditional processed product of Pinellia ternata (Thunb.) Berit., which mainly used for treating cold asthma (CA). However, the mechanism of action of PRP for treating CA have not been fully elucidated. AIM OF THE STUDY: To investigate the core active constituents and the pharmacological mechanism of PRP against CA. MATERIALS AND METHODS: Ovalbumin (OVA) and cold water-induced cold asthma model were established in male mice. The effects of water extract from PRP were evaluated by general morphological observation, expectorant activity, airway hyperresponsiveness, mucus hypersecretion, inflammatory cytokines, etc. Additionally, the mRNA and protein expression of mucin 5AC (MUC5AC) and aquaporin 5 (AQP5) in vivo and in vitro were detected by immunohistochemistry (IHC), qRT-PCR, and western blotting. The mechanisms of action were investigated through network pharmacology and transcriptomic, and validated through western blotting and molecular docking. RESULTS: PRP exhibited a favorable expectorant activity, and significantly reduced the airway inflammation, mucus secretion, and hyperresponsiveness in cold asthma model. It also reduced the levels of IL-4, IL-5, IL-8, and IL-13 in bronchoalveolar lavage fluid (BALF) and IL-4 and total IgE in serum, while obviously increased the levels of IL-10 and IFN-γ in serum for asthmatic mice. Meanwhile, PRP also attenuated the pathological changes and mucus production in cold asthmatic mice. Moreover, the downregulation of MUC5AC and upregulation of AQP 5 were detected by western blotting and qRT-PCR after administration with PRP both in vivo and in vitro. PRP expectedly inhibited the protein expression of PKC-α, SRC, p-EGFR, p-ERK1/2, p-JNK, p-p38, p-PI3K, and p-Akt levels in vivo. CONCLUSIONS: These combined data showed that PRP suppressed the allergic airway inflammation of CA by regulating the balance of Th1 and Th2 cytokines and the possible involvement of the PKC/EGFR/MAPK/PI3K-Akt signaling pathway. Pentadecanoic acid, licochalcone A, ß-sitosterol, etc. were considered as main active ingredients of PRP against CA. This study provides a novel perspective of the classical herbal processed product PRP in the treatment of CA.

Protective effect of Amomum Roxb. essential oils in lipopolysaccharide-induced acute lung injury mice and its metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Several Amomum species are commonly used in food as flavoring agents and traditional Chinese medicine to treat inflammation-related diseases. AIM OF THE STUDY: This study aims to investigate the protective effects of Chinese herbal medicines, including six Amomum Roxb. essential oils (AEOs), against acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. MATERIALS AND METHODS: The compositions of AEOs were analyzed using gas chromatography - mass spectrometry. RAW264.7 cells were treated with AEOS (0-100 µg/mL) and stimulated with LPS. C57 mice received AEOs (100 mg/kg) via atomization system for seven consecutive days, and then, intratracheal instillation of LPS was applied to establish an in vivo model of acute lung injury. RESULTS: We identified three AEOs demonstrating anti-inflammatory effects and amelioration of LPS-induced lung tissue pathological damage. Furthermore, we found that these AEOs reduced lung wet/dry weight ratios and protein concentrations in the bronchoalveolar lavage fluid of mice with LPS-induced ALI. Additionally, AEOs reduced the levels of malondialdehyde, TNF-α, IL-6, and IL-1ß but increased the levels of superoxide dismutase and catalase in lung tissue, alveolar lavage fluid, and serum samples. We also found that these three AEOs affected proteins related to the TLR4/Myd88/NF-κB pathway. CONCLUSIONS: In summary, our findings revealed that AEOs ameliorate inflammatory and oxidative stress in mice with ALI through the TLR4/Myd88/NF-κB pathway.

Single Treatment of Vitamin D3 Ameliorates LPS-Induced Acute Lung Injury through Changing Lung Rodentibacter abundance.

Acute lung injury (ALI) is characterized by severe inflammation. Vitamin D3 is discussed to reduce inflammation in ALI, but the mechanism is not well understood. This study assesses the effect of different calcitriol administration strategies on inflammation and the lung microbiota composition in ALI. In a mouse model, the alveolus and airway pathology are assessed by immunohistology. mRNA expression is determined by Real-Time Quantitative PCR and protein expressions is detected by Western-blotting. The composition of microbiota is performed by 16s DNA high-throughput sequencing. Short-term vitamin D3 supplementation prevents lipopolysaccharide-induced ALI by preventing pro-inflammatory cytokines including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). In contrast, long-term treatment over 3 days, 6 days, or 10 days had no such effect. Short-term vitamin D3, but not long-term pretreatment significantly reduces the phosphorylation of signal transducer and activator of transcription 3 and suppressor of cytokine signaling 3, but upregulates the phosphorylation of inhibitor of nuclear factor-κ-gene binding. Furthermore, an increased relative abundance of Rodentibacter genus in LPS-challenged mice bronchoalveolar lavage fluid is observed, which is sensitive to short-term vitamin D3 treatment, effectively alleviating the Rodentibacter abundance. Correlation analysis shows that the load of Rodentibacter positively correlated with the IL-1ß, IL-6, and TNF-α gene expression. The data support that a single administration of vitamin D3 may work as an adjuvant therapy for acute lung inflammation.

The efficacy and safety of pinocembrin in a sheep model of bleomycin-induced pulmonary fibrosis.

The primary flavonoid, pinocembrin, is thought to have a variety of medical uses which relate to its reported anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer properties. Some studies have reported that this flavonoid has anti-fibrotic activities. In this study, we investigated whether pinocembrin would impede fibrosis, dampen inflammation and improve lung function in a large animal model of pulmonary fibrosis. Fibrosis was induced in two localized lung segments in each of the 10 sheep participating in the study. This was achieved via two infusions of bleomycin delivered bronchoscopically at a two-week interval. Another lung segment in the same sheep was left untreated, and was used as a healthy control. The animals were kept for a little over 5 weeks after the final infusion of bleomycin. Pinocembrin, isolated from Eucalyptus leaves, was administered to one of the two bleomycin damaged lung segments at a dose of 7 mg. This dose was given once-weekly over 4-weeks, starting one week after the final bleomycin infusion. Lung compliance (as a measure of stiffness) was significantly improved after four weekly administrations of pinocembrin to bleomycin-damaged lung segments. There were significantly lower numbers of neutrophils and inflammatory cells in the bronchoalveolar lavage of bleomycin-infused lung segments that were treated with pinocembrin. Compared to bleomycin damaged lung segments without drug treatment, pinocembrin administration was associated with significantly lower numbers of immuno-positive CD8+ and CD4+ T cells in the lung parenchyma. Histopathology scoring data showed that pinocembrin treatment was associated with significant improvement in inflammation and overall pathology scores. Hydroxy proline analysis showed that the administration of pinocembrin did not reduce the increased collagen content that was induced by bleomycin in this model. Analyses of Masson's Trichrome stained sections showed that pinocembrin treatment significantly reduced the connective tissue content in lung segments exposed to bleomycin when compared to bleomycin-infused lungs that did not receive pinocembrin. The striking anti-inflammatory and modest anti-fibrotic remodelling effects of pinocembrin administration were likely linked to the compound's ability to improve lung pathology and functional compliance in this animal model of pulmonary fibrosis.

Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) turn out to be a promising source of cell-free therapy. Here, we investigated the biodistribution and effect of nebulized human adipose-derived MSC-EVs (haMSC-EVs) in the preclinical lung injury model and explored the safety of nebulized haMSC-EVs in healthy volunteers. DiR-labelled haMSC-EVs were used to explore the distribution of nebulized haMSC-EVs in the murine model. Pseudomonas aeruginosa-induced murine lung injury model was established, and survival rate, as well as WBC counts, histology, IL-6, TNF-α and IL-10 levels in bronchoalveolar lavage fluid (BALF) were measured to explore the optimal therapeutic dose of haMSC-EVs through the nebulized route. Twenty-four healthy volunteers were involved and received the haMSC-EVs once, ranging from 2 × 108 particles to 16 × 108 particles (MEXVT study, NCT04313647). Nebulizing haMSC-EVs improved survival rate to 80% at 96 h in P. aeruginosa-induced murine lung injury model by decreasing lung inflammation and histological severity. All volunteers tolerated the haMSC-EVs nebulization well, and no serious adverse events were observed from starting nebulization to the 7th day after nebulization. These findings suggest that nebulized haMSC-EVs could be a promising therapeutic strategy, offering preliminary evidence to promote the future clinical applications of nebulized haMSC-EVs in lung injury diseases.

In vitro and in vivo relaxation and anti-inflammation of natural flavonoids from Elaeagnus pungens leaf via L-type calcium channel and targeting MAPK signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), the leaf of Elaeagnus pungens Thunb. (Family Elaeagnaceae) is a herb documented as an antiasthmatic remedy to treat the severe asthma, bronchitis and other respiratory diseases in the early material medica "Bencao Gangmu" (Ming dynasty, about 442 years ago). AIM OF THE STUDY: This work is purposed to investigate the pharmacological effects and mechanism of total flavonoids from Elaeagnus pungens leaves (FLA) on asthma in vivo and vitro. MATERIALS AND METHODS: Female BALB/c mice were sensitized by intraperitoneal injection of OVA with aluminum hydroxide and intranasal challenged with OVA. After treatment with FLA (10, 20 mg/kg p.o.), the behaviors of mice were observed by score evaluation. Enumeration of total cells and OVA-specific IgE assay in the blood were measured as well as enumeration of total cells and cytokines assay in the BALF. Furthermore, histopathological analysis was performed by HE staining. The in vitro relaxing action on muscle force of FLA (0.0316-10.0 mg/ml) was evaluated using isometric tension in tracheal rings, and VDLCC currents were recorded to explore the relaxation mechanism in the isolated tracheal rings and mouse ASM cells, respectively. In vitro anti-inflammatory actions were assessed with LPS-stimulated RAW 264.7 macrophages. The production of inflammatory mediators and MAPK signaling pathway was estimated using ELISA and Western blotting analysis, respectively. RESULTS: The high dose of flavones from E. pungens leaf (20 mg/kg) can significantly improve the symptom of asthma breakout and relieve the lung swelling. FLA treatment decreased eosinophils and leukocytes numbers in blood and BLAF with a dosedependent manner. Furthermore, the inhibiting effect of FLA on the level of Ig E and inflammatory-related cytokines including TNF-α, IL-5 showed dose-independent. FLA relaxed high K + -induced contraction in a dose-dependent manner. The maximal relaxation produced by FLA was 99.7% (IC 50 = 0.46 mg/ml). The whole-cell VDLCC currents were abolished by FLA (3.16 mg/ml) and FLA significantly decreased the maximal amplitude of VDLCCs. No cytotoxic effect of FLA was observed in RAW264.7 cells under the tested concentrations (1-300 µg/mL). The increased IL-6 and NO by the stimulation of LPS in RAW264.7 cells were significantly inhibited by FLA in the dosedependent manner. Treatment with LPS in the presence of FLA, LPS-induced phosphorylation of ERK1/2 and JNK was inhibited in the macrophages. CONCLUSION: FLA from Elaeagnus pungens leaf can alleviate the inflammation symptom via reducing the eosinophils and leukocytes numbers as well as the production of pro-inflammatory cytokines. This anti-inflammatory effect is related to the modulation of the MAPK signaling pathway. FLA can relax the precontracted TRs by blocking the VDLCCs, which interrupts extracellular Ca 2+ influx and inhibit the rise of [Ca 2+ ]i. It strongly suggests that these flavonoids components are the substances basis of Elaeagnus pungens leaves for allergic action, bronchospasm and inflammation in asthma.

Comparison of plasma and pulmonary availability of chlorogenic acid, forsythiaside A and baicalin after intratracheal and intravenous administration of Shuang-Huang-Lian injection.

ETHNOPHARMACOLOGICAL RELEVANCE: The off-label nebulization of Shuang-Huang-Lian (SHL) injection is often utilized to treat respiratory tract infections in China. However, the pulmonary biopharmaceutics of SHL was generally unknown, limiting the rational selection of therapeutic dose and dose frequency. AIM OF THE STUDY: To characterize the size distribution of nebulized aerosols and to compare the pharmacokinetics and the lung distribution of three chemical makers of SHL, chlorogenic acid (CHA), forsythiaside A (FTA) and baicalin (BC), after intratracheal and intravenous administration of SHL to rats. MATERIALS AND METHODS: The droplet size distribution profiles over nebulization process were dynamically monitored using a laser diffraction method whereas the levels of CHA, FTA and BC in plasma, lung tissues and bronchoalveolar lavage fluids (BALF) were determined by a validated LC-MS/MS assay. The pulmonary anti-inflammatory efficacy was evaluated using a lipopolysaccharide (LPS) induced lung inflammation model as indicated by the level of tumor necrosis factor-α (TNF-α) in BALF. RESULTS: The nebulization of SHL showed good inhalability and allowed the aerosols to reach the upper or lower respiratory tract dependent on the performance of selected nebulizers. Following intratracheal administration of SHL at different doses, CHA, FTA and BC were absorbed into the bloodstream with the mean absorption time being 67.5, 63.5 and 114 min, respectively, rendering mean absolute bioavailabilities between 42.4% and 61.4% roughly independent of delivered dose. Relative to the intravenous injection, the intrapulmonary delivery increased the lung-to-plasma concentration ratios of CHA, FTA and BC by more than 100 folds and markedly improved the lung availability by 563-676 folds, leading to enhanced and prolonged lung retention. The production of TNF-α in BALF was decreased by ~50% at an intratracheal dose of 125 µL/kg SHL to LPS-treated mice. CONCLUSION: The nebulization delivery of SHL is a promising alternative to the intravenous injection for the treatment of respiratory tract infections.

Electroacupuncture inhibits IL-17/IL-17R and post-receptor MAPK signaling pathways in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Interleukin (IL)-17, as a T-helper 17 cell (Th17) cytokine, plays a key role in chronic obstructive pulmonary disease (COPD) pathophysiology including chronic inflammation and airway obstruction, which lead to decreased pulmonary function. The aim of this study was to investigate the effect of acupuncture on IL-17, its receptor (IL-17R) and the mitogen-activated protein kinase (MAPK) signaling pathway, in a rat model of COPD. METHODS: The COPD model was induced in Sprague Dawley rats by exposure to cigarette smoke for 12 weeks. The model rats were treated with electroacupuncture (EA) at BL13 and ST36. The lung function and histology of the rats were observed. IL-17, tumor necrosis factor (TNF)-α, and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and in plasma. The leukocytes and macrophages in the BALF were counted. The expression levels of IL-17R were assayed in lung tissue by real-time polymerase chain reaction (PCR), western blotting, and immunohistochemistry. MAPK signaling pathway molecules including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38, and their phosphorylated forms, were observed in the lung by western blotting. RESULTS: Compared with the control group rats, lung function decreased and there was a severe inflammatory infiltration of the pulmonary parenchyma in the COPD rats. EA effectively improved lung function and alleviated the inflammatory infiltration in the lungs of COPD rats. EA also reversed the elevated total leukocyte and macrophage counts, the high levels of IL-17 and TNF-α, and the low IL-10 content in COPD rats. Meanwhile, EA downregulated the increased mRNA and protein expression of IL-17R, and significantly inhibited the elevated levels of phosphorylated JNK, ERK1/2, and p38 in the lungs of COPD rats. CONCLUSION: Our results suggest that the protective effects of acupuncture therapy on the lungs of COPD rats are likely related to inhibition of IL-17/IL-17R and the post-receptor MAPK signaling pathways.

Ferulic acid ameliorates the progression of pulmonary fibrosis via inhibition of TGF-ß/smad signalling.

Natural products are one of the best sources for the discovery of novel drugs and compounds for multiple diseases. Pulmonary fibrosis (PF) is a chronic, progressive, irreversible, and fatal fibrotic disorder of lungs with unknown etiology and finite therapeutic choices. The use of naturally occurring phytomedicines has emerged to counteract many fibrotic disorders involving oxidative stress and inflammation. In the present study, we evaluated the protective effects of ferulic acid (FA), in an animal model of silica-induced PF. Pulmonary function of mice was evaluated by performing radiological analysis, bronchoalveolar lavage fluid (BALF), inflammatory cytokines, histology and protein expression studies. Our findings revealed that mice challenged with silica displayed characteristic features of pulmonary injury and fibrosis. However, treatment with FA significantly restored the accumulation of inflammatory cells in BALF. FA led to a partial reversal of silica-induced fibrotic changes in the pulmonary tissue. Subsequently, FA halts the progression of PF in a dose-dependent manner by ameliorating the expression of fibrotic proteins including collagen-I, TGF-ß, p-smad2/3 and prevented epithelial-mesenchymal transition (EMT). Collectively, the present study suggests that the inhibition of oxidative stress, inflammatory and TGF-ß/smad signalling might be involved in the observed anti-fibrotic benefits of FA against silica-induced PF in mice.

Silica Induction of Diverse Inflammatory Proteome in Lungs of Lupus-Prone Mice Quelled by Dietary Docosahexaenoic Acid Supplementation.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1ß, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA's effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE-mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.

The effect of Zataria multiflora hydroalcoholic extract on memory and lung changes induced by rats that inhaled paraquat.

Objectives: The effects of hydroalcoholic extract of Zataria multiflora (Z. multiflora) on memory changes, as well as lung injury due to inhaled paraqut (PQ) in rat, were examined.Method: Control group of rat with saline aerosol administration, PQ groups with PQ aerosol (27 and 54 mg/m3) administration, PQ groups treated with two doses of the extract (200 and 800 mg/kg/day) and dexamethasone (0.03 mg/kg/day) were studied. Shuttle box and Morris Water Maze (MWM) tests were carried out as well as oxidant, anti-oxidant markers, total and differential white blood cell (WBC) counts and cytokine levels in broncho-alveolar lavage (BALF).Results: Inhaled PQ significantly increased the escape latency and travelled distance in MWM test, but the time spent in the target quadrant on the probe day was significantly reduced (p < 0.05 to p < 0.001). The latency to enter the dark room at 3, 24, and 48 h after an electrical shock was reduced due to PQ (p < 0.05 to p < 0.001). Exposure to PQ significantly increased total WBC, neutrophil, eosinophil, lymphocyte, and monocyte counts, IL-10, interferon gama (INF-γ), nitrite (NO2), and malondialdehyde (MDA) levels, but catalase (CAT), superoxide dismutase (SOD), and thiol levels were decreased (p < 0.05 to p < 0.00). Z. multiflora and dexamethasone treatment significantly improved all behavioral as well as lung changes induced by inhaled PQ (p < 0.05 to p < 0.01).Conclusion: Z. multiflora treatment improved learning and memory impairment as well as lung inflammation and oxidative stress induced by inhaled PQ.

Methods for Experimental Allergen Immunotherapy: Subcutaneous and Sublingual Desensitization in Mouse Models of Allergic Asthma.

Allergic asthma is characterized by airway hyperresponsiveness, remodeling, and reversible airway obstruction. This is associated with an eosinophilic inflammation of the airways, caused by inhaled allergens such as house dust mite or grass pollen. The inhaled allergens trigger a type-2 inflammatory response with the involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high immunoglobulin E (IgE) antibody production by B cells and mucus production by airway epithelial cells. As a consequence of the IgE production, subsequent allergen reexposure results in a classic allergic response with distinct early and late phases, both resulting in bronchoconstriction and shortness of breath. Allergen-specific immunotherapy (AIT) is the only treatment that is capable of modifying the immunological process underlying allergic responses including allergic asthma. Both subcutaneous AIT (SCIT) as well as sublingual AIT (SLIT) have shown clinical efficacy in long-term suppression of the allergic response. Although AIT treatments are very successful for rhinitis, application in asthma is hampered by variable efficacy, long duration of treatment, and risk of severe side effects. A more profound understanding of the mechanisms by which AIT induces tolerance to allergens in sensitized individuals is needed to be able to improve its efficacy. Mouse models have been very valuable in preclinical research for characterizing the mechanisms of desensitization in AIT and evaluating novel approaches to improve its efficacy. Here, we present a rapid and reproducible mouse model for allergen-specific immunotherapy. In this model, mice are sensitized with two injections of allergen adsorbed to aluminum hydroxide, followed by subcutaneous injections (SCIT) or sublingual administrations (SLIT) of allergen extracts as an immunotherapy treatment. Finally, mice are challenged by intranasal allergen administrations. We will also describe the protocols as well as the most important readout parameters for the measurements of invasive lung function, serum immunoglobulin levels, isolation of bronchoalveolar lavage fluid (BALF), and preparation of cytospin slides. Moreover, we describe how to perform ex vivo restimulation of lung single-cell suspensions with allergens, flow cytometry for identification of relevant immune cell populations, and ELISAs and Luminex assays for assessment of the cytokine concentrations in BALF and lung tissue.

Proteomic and mechanistic study of Qingxuan Tongluo formula and curcumin in the treatment of Mycoplasma pneumoniae pneumonia.

OBJECTIVE: Mycoplasma pneumoniae (MP) is the only pathogen in the Mycoplasma family that can cause respiratory symptoms, including acute upper respiratory tract infection and bronchitis, which are often attributed to Mycoplasma pneumoniae pneumonia (MPP). MPP is one of the diseases that commonly affects the pediatric respiratory system, but its pathogenesis is unclear. This study investigated the therapeutic effects and mechanisms of Qingxuan Tongluo formula and its main component, curcumin, on MPP. METHODS: A mouse model of MPP was obtained by nasal drip of the MP strain. The effects of Qingxuan Tongluo formula and curcumin on the treatment of MPP were studied. The proteomic profiles of the alveolar lavage fluid of mice in the model group, Qingxuan Tongluo formula group and curcumin group were evaluated by LC-MS/MS. ELISA and immunohistochemistry were used to verify the possible presence of MP infection biomarkers and drug target proteins. RESULTS: Compared with the mice in the model group, the MPP mice in the Qingxuan Tongluo formula group had significantly reduced fever and cough and prolonged the cough incubation period. Moreover, the pulmonary pathology of the MPP mice was significantly improved, and the lung histopathological score was decreased. After treatment with Qingxuan Tongluo formula and curcumin, the functional and pathway abnormalities caused by MP were mainly inhibited. Levels of HSP90AA1, GRP94, ENO1 and PLG expression were verified by ELISA and immunohistochemistry. CONCLUSION: Qingxuan Tongluo formula significantly reduced fevers and cough and prolonged the cough incubation period of MPP mice. Qingxuan Tongluo formula and curcumin significantly improved the pathological changes in lung tissue caused by MP infection. Proteomics analyses indicated that Qingxuan Tongluo formula and curcumin may have therapeutic effects on MPP by regulating energy metabolism, relieving oxidative stress and activating the fibrinolytic system. ENO1 and PLG were found to be potential drug targets.

Mushroom Inonotus sanghuang alleviates experimental pulmonary fibrosis: Implications for therapy of pulmonary fibrosis.

Mushroom Inonotus sanghuang has been characterized as a traditional medicine in China and has pharmacological activities to treat inflammation, gastroenteric dysfunction, and cancer. Recently, we reported the impact of Inonotus sanghuang extract (ISE) from ethyl acetate fraction on bleomycin (BLM)-induced acute lung injury in mice. Here, we aimed to investigate ISE's impact on pulmonary fibrosis using in vivo and in vitro models and the underlying mechanisms. To evaluate pulmonary fibrosis, female C57BL/6 mice fed ISE (0% or 0.6% in diet) for 4 weeks were instilled intratracheally with BLM and then continued the same diet before the end of the experiment. A549 cells were used to evaluate the epithelial-mesenchymal transition (EMT). Feeding ISE improved BLM-treated mice's survival via decreasing lung infiltrating cells and fibrosis, followed by reducing hydroxyproline content, collagen deposition, and mesenchymal markers (α-SMA and vimentin) while increasing epithelial marker E-cadherin. ISE also suppressed the TGF-ß expression, Smad2/3 phosphorylation, and EMT-related transcription factor Snail upon BLM instillation. Iin vitro study demonstrated that ISE inhibited TGF-ß-induced EMT-like phenotype and cell behaviors, the expression of α-SMA and vimentin, and prevented E-cadherin reduction of A549 cells. Consistent with in vivo study, ISE abrogated p-Smad2/3, and Snail expression. Finally, the influence of ISE on EMT was not due to ISE toxicity. Our findings indicated that ISE effectively attenuated BLM-induced lung fibrosis. These ISE properties were thought to be involved in interfering TGF-ß, Smad2/3 phosphorylation, and EMT process, suggesting that the material has the potential health benefits to improve lung fibrosis.

Cardiopulmonary function and dysregulated cardiopulmonary reflexes following acute oleoresin capsicum exposure in rats.

Cardiopulmonary functions such as respiratory depression, severe irritation, inflamed respiratory tract, hyperventilation and, tachycardia are the most affected ones when it comes to the riot control agent oleoresin capsicum (OC) exposure. However, no studies have been done to elucidate the mechanism underlying deterioration of the combined cardiopulmonary functions. Parameters such as acute respiratory, cardiac, parameters and ultrasonography (USG) measurements were investigated in an in vivo setup using Wistar rats at 1 h and 24 h post inhalation exposure to 2%, 6% and 10% OC, whereas, cell migration in rat peritoneal mast cells (RPMCs), metabolomics and eosinophil peroxidase (EPO) activity in bronchoalveolar lavage fluid (BALF) were investigated in an in vitro setup. Results obtained from electrophysiological recording indicated that OC exposure produces apnea and decrease in mean arterial pressure (MAP) was obtained from hemodynamic parameters whereas cardiac parameters assessment revealed increase in the level of cardiac output (CO) and decrease in stroke volume (SV) with recovery towards the post-exposure period. A decrease in the percentage area of certain fatty acid pathway metabolites in BALF appropriately linked the lung injury following OC exposure which was further cemented by increasing concentration of EPO. Histopathology and SEM also proved to be favorable techniques for the detection of OC induced physiological cardiac and pulmonary modifications respectively. Furthermore, Boyden chamber experiment established the chemoattractant property of OC. It may be concluded from the above studies that these newly reported facets may be utilized pharmacologically to mitigate cardiopulmonary adverse effects owing to OC exposure.

Novel methods for the analysis of toxicants in bronchoalveolar lavage fluid samples from e-cigarette, or vaping, product use-associated lung injury cases: Selected petroleum distillates.

RATIONALE: Over 2700 e-cigarette, or vaping, product use-associated lung injury (EVALI) cases were reported to the Centers for Disease Control and Prevention (CDC) during August 2019-February 2020. Bronchoalveolar lavage (BAL) fluid samples from 51 EVALI and 99 non-EVALI cases were analyzed for toxicants including petroleum distillates. We describe a novel method to measure petroleum distillates in BAL fluid using gas chromatography-mass spectrometry (GC/MS). METHODS: n-Hexane, n-heptane, n-octane, methylcyclopentane, and cyclohexane were measured in BAL fluid specimens by headspace solid-phase microextraction/GC/MS. We created and characterized BAL fluid pools from non-EVALI individuals to determine assay accuracy, precision, linearity, limits of detection (LODs), and analytical specificity. All measurements were conducted in accordance with the rigorous method validation procedures of CDC's Division of Laboratory Sciences. RESULTS: Matrix validation experiments showed that calibration curves in BAL fluid and saline had similar slopes, with differences less than 5%. Assay precision ranged from 1.98% to 18%. In addition, the LODs for the five analytes ranged from 0.05 to 0.10 µg/L, and their linearity was confirmed with R2 values >0.99. The analysis of selected petroleum distillates in BAL fluid analysis was shown to be comparable with their analysis in blood in which the 95th percentiles are below detection. CONCLUSIONS: We developed and validated a method to quantify petroleum distillates in BAL fluid specimens using GC/MS. The assay provided precise and accurate analyses of EVALI and non-EVALI BAL fluid specimens in support of CDC's EVALI response. This method is applicable to the determination of a broad range of volatile organic compounds in BAL fluid specimens.

Osthole attenuates ovalbumin­induced lung inflammation via the inhibition of IL­33/ST2 signaling in asthmatic mice.

Asthma is a common chronic inflammatory airway disease. Recent studies have reported that interleukin (IL)­33 is a potential link between the airway epithelium and Th2­type inflammatory responses, which are closely related to the progression of asthma. The IL­33 receptor, ST2, is highly expressed in group 2 innate lymphoid cells (ILC2s), Th2 cells, mast cells, eosinophils and natural killer (NK) cells. Cnidii Fructus is a Chinese herb with a long history of use in the treatment of asthma in China. Osthole is one of the major components of Cnidii Fructus. The present study examined the anti­asthmatic effects of osthole in mice and aimed to elucidate the underlying mechanisms involving the IL­33/ST2 pathway. BALB/c mice were sensitized and challenged with ovalbumin and then treated with an intraperitoneal injection of osthole (25 and 50 mg/kg). Subsequently, the airway hyper­responsiveness (AHR) and inflammation of the lungs were evaluated. The amounts of IL­4, IL­5, IL­13, interferon (IFN)­Î³ and IL­33 in the bronchoalveolar lavage fluid (BALF) were measured by Luminex assay and their mRNA levels in the lungs were measured by reverse transcription­quantitative PCR. The histopathology of the lungs was performed with H&E, PAS and Masson's staining. The expression of ST2 in the lungs was evaluated by immunohistochemistry. The data demonstrated that osthole markedly reduced AHR and decreased the number of eosinophils and lymphocytes in BALF. It was also observed that osthole significantly inhibited the release of Th2­type cytokines (IL­4, IL­5 and IL­13) and upregulated the IFN­Î³ level in BALF. Moreover, osthole significantly attenuated the IL­33 and ST2 expression in the lungs of asthmatic mice. On the whole, osthole attenuated ovalbumin­induced lung inflammation through the inhibition of IL­33/ST2 signaling in an asthmatic mouse model. These results suggest that osthole is a promising target for the development of an asthma medication.