Role of macrophage bioenergetics in N-acetylcysteine-mediated mitigation of lung injury and oxidative stress induced by nitrogen mustard.

Nitrogen mustard (NM) is a toxic vesicant that causes acute injury to the respiratory tract. This is accompanied by an accumulation of activated macrophages in the lung and oxidative stress which have been implicated in tissue injury. In these studies, we analyzed the effects of N-acetylcysteine (NAC), an inhibitor of oxidative stress and inflammation on NM-induced lung injury, macrophage activation and bioenergetics. Treatment of rats with NAC (150 mg/kg, i.p., daily) beginning 30 min after administration of NM (0.125 mg/kg, i.t.) reduced histopathologic alterations in the lung including alveolar interstitial thickening, blood vessel hemorrhage, fibrin deposition, alveolar inflammation, and bronchiolization of alveolar walls within 3 d of exposure; damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage fluid protein and cells, was also reduced by NAC, along with oxidative stress as measured by heme oxygenase (HO)-1 and Ym-1 expression in the lung. Treatment of rats with NAC attenuated the accumulation of macrophages in the lung expressing proinflammatory genes including Ptgs2, Nos2, Il-6 and Il-12; macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)α protein were also reduced in histologic sections. Conversely, NAC had no effect on macrophages expressing the anti-inflammatory proteins arginase-1 or mannose receptor, or on NM-induced increases in matrix metalloproteinase (MMP)-9 or proliferating cell nuclear antigen (PCNA), markers of tissue repair. Following NM exposure, lung macrophage basal and maximal glycolytic activity increased, while basal respiration decreased indicating greater reliance on glycolysis to generate ATP. NAC increased both glycolysis and oxidative phosphorylation. Additionally, in macrophages from both control and NM treated animals, NAC treatment resulted in increased S-nitrosylation of ATP synthase, protecting the enzyme from oxidative damage. Taken together, these data suggest that alterations in NM-induced macrophage activation and bioenergetics contribute to the efficacy of NAC in mitigating lung injury.

Inhaled paraquat-induced lung injury in rat, improved by the extract of Zataria multiflora boiss and PPARγ agonist, pioglitazone.

The effects of a PPAR-γ agonist, pioglitazone and Zataria multiflora (Z. multiflora) on inhaled paraquat (PQ)-induced lung oxidative stress, inflammation, pathological changes and tracheal responsiveness were examined. The study was carried out in control rats exposed to normal aerosol of saline, PQl and PQh groups exposed to aerosols of 27 and 54 mg/m3 PQ, groups exposed to high PQ concentration (PQh) and treated with 200 and 800 mg/kg/day Z. multiflora, 5 and 10 mg/kg/day pioglitazone, low doses of Z. multiflora + pioglitazone, and 0.03 mg/kg/day dexamethasone. Increased tracheal responsiveness, transforming growth factor beta (TGF-ß) and lung pathological changes due to PQh were significantly improved by high doses of Z. multiflora and pioglitazone, dexamethasone and extract + pioglitazone, (p < 0.05 to p < 0.001). In group treated with low doses of the extract + pioglitazone, the improvements of most measured variables were significantly higher than the low dose of two agents alone (p < 0.05 to p < 0.001). Z. multiflora improved lung injury induced by inhaled PQ similar to dexamethasone and pioglitazone which could be mediated by PPAR-γ receptor.

Adropin attenuates pancreatitis­associated lung injury through PPARγ phosphorylation­related macrophage polarization.

Acute pancreatitis (AP)­associated lung injury (ALI) is a critical complication of AP. Adropin is a regulatory protein of immune metabolism. The present study aimed to explore the immunomodulatory effects of adropin on AP­ALI. For this purpose, serum samples of patients with AP were collected and the expression levels of serum adropin were detected using ELISA. Animal models of AP and adropin knockout (Adro­KO) were constructed, and adropin expression in serum and lung tissues was investigated. The levels of fibrosis and apoptosis were evaluated using hematoxylin and eosin staining, Masson's staining and immunohistochemistry of in lung tissue. M1/M2 type macrophages in the lungs were detected using immunofluorescence staining, western blot analysis and reverse transcription­quantitative PCR. As shown by the results, adropin expression was decreased in AP. In the Adro­KO + L­arginine (L­Arg) group, macrophage infiltration, fibrosis and apoptosis were increased. The expression of peroxisome proliferator­ activated receptor γ (PPARγ) was downregulated, and the macrophages exhibited a trend towards M1 polarization in the Adro­KO + L­Arg group. Adropin exogenous supplement attenuated the levels of fibrosis and apoptosis in the model of AP. Adropin exogenous supplement also increased PPARγ expression by the regulation of the phosphorylation levels, which was associated with M2 macrophage polarization. On the whole, the findings of the present study suggest that adropin promotes the M2 polarization of lung macrophages and reduces the severity of AP­ALI by regulating the function of PPARγ through the regulation of its phosphorylation level.

Pi-Pa-Run-Fei-Tang alleviates lung injury by modulating IL-6/JAK2/STAT3/IL-17 and PI3K/AKT/NF-κB signaling pathway and balancing Th17 and Treg in murine model of OVA-induced asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Pi-Pa-Run-Fei-Tang (PPRFT) is an empirical TCM prescription for treating asthma. However, the underlying mechanisms of PPRFT in asthma treatment have yet to be elucidated. Recent advances have revealed that some natural components could ameliorate asthma injury by affecting host metabolism. Untargeted metabolomics can be used to better understand the biological mechanisms underlying asthma development and identify early biomarkers that can help advance treatment. AIM OF THE STUDY: The aim of this study was to verification the efficacy of PPRFT in the treatment of asthma and to preliminarily explore its mechanism. MATERIALS AND METHODS: A mouse asthma model was built by OVA induction. Inflammatory cell in BALF was counted. The level of IL-6, IL-1ß, and TNF-α in BALF were measured. The levels of IgE in the serum and EPO, NO, SOD, GSH-Px, and MDA in the lung tissue were measured. Furthermore, pathological damage to the lung tissues was detected to evaluate the protective effects of PPRFT. The serum metabolomic profiles of PPRFT in asthmatic mice were determined by GC-MS. The regulatory effects on mechanism pathways of PPRFT in asthmatic mice were explored via immunohistochemical staining and western blotting analysis. RESULTS: PPRFT displayed lung-protective effects through decreasing oxidative stress, airway inflammation, and lung tissue damage in OVA-induced mice, which was demonstrated by decreasing inflammatory cell levels, IL-6, IL-1ß, and TNF-α levels in BALF, and IgE levels in serum, decreasing EPO, NO, and MDA levels in lung tissue, elevating SOD and GSH-Px levels in lung tissue and lung histopathological changes. In addition, PPRFT could regulate the imbalance in Th17/Treg cell ratios, suppress RORγt, and increase the expression of IL-10 and Foxp3 in the lung. Moreover, PPRFT treatment led to decreased expression of IL-6, p-JAK2/Jak2, p-STAT3/STAT3, IL-17, NF-κB, p-AKT/AKT, and p-PI3K/PI3K. Serum metabolomics analysis revealed that 35 metabolites were significantly different among different groups. Pathway enrichment analysis indicated that 31 pathways were involved. Moreover, correlation analysis and metabolic pathway analysis identified three key metabolic pathways: galactose metabolism; tricarboxylic acid cycle; and glycine, serine, and threonine metabolism. CONCLUSION: This research indicated that PPRFT treatment not only attenuates the clinical symptoms of asthma but is also involved in regulating serum metabolism. The anti-asthmatic activity of PPRFT may be associated with the regulatory effects of IL-6/JAK2/STAT3/IL-17 and PI3K/AKT/NF-κB mechanistic pathways.

Prenatal vitamin D supplementation mitigates inflammation-related alveolar remodeling in neonatal mice.

The development of chronic lung disease in the neonate, also known as bronchopulmonary dysplasia (BPD), is the most common long-term complication in prematurely born infants. In BPD, the disease-characteristic inflammatory response culminates in nonreversible remodeling of the developing gas exchange area, provoked by the impact of postnatal treatments such as mechanical ventilation (MV) and oxygen treatment. To evaluate the potential of prenatal treatment regimens to modulate this inflammatory response and thereby impact the vulnerability of the lung toward postnatal injury, we designed a multilayered preclinical mouse model. After administration of either prenatal vitamin D-enriched (VitD+; 1,500 IU/g food) or -deprived (VitD-; <10 IU/kg) food during gestation in C57B6 mice (the onset of mating until birth), neonatal mice were exposed to hyperoxia (FiO2 = 0.4) with or without MV for 8 h at days 5-7 of life, whereas controls spontaneously breathed room air. Prenatal vitamin D supplementation resulted in a decreased number of monocytes/macrophages in the neonatal lung undergoing postnatal injury together with reduced TGF-ß pathway activation. In consequence, neonatal mice that received a VitD+ diet during gestation demonstrated less extracellular matrix (ECM) remodeling upon lung injury, reflected by the reduction of pulmonary α-smooth muscle actin-positive fibroblasts, decreased collagen and elastin deposition, and lower amounts of interstitial tissue in the lung periphery. In conclusion, our findings support strategies that attempt to prevent vitamin D insufficiency during pregnancy as they could impact lung health in the offspring by mitigating inflammatory changes in neonatal lung injury and ameliorating subsequent remodeling of the developing gas exchange area.NEW & NOTEWORTHY Vitamin D-enriched diet during gestation resulted in reduced lung inflammation and matrix remodeling in neonatal mice exposed to clinically relevant, postnatal injury. The results underscore the need to monitor the subclinical effects of vitamin D insufficiency that impact health in the offspring when other risk factors come into play.

Potential of hydroethanolic leaf extract of Ocimum sanctum in ameliorating redox status and lung injury in COPD: an in vivo and in silico study.

Oxidative stress and inflammation are hypothesised as the main contributor for Chronic Obstructive Pulmonary Disease (COPD). Cigarette smoke (CS), a major cause of COPD leads to inflammation resulting in recruitment of neutrophils and macrophages which are rich sources of oxidants. Activation of these cells produces excess oxidants and depletes antioxidants resulting in stress. Presently, effective drug for COPD is limited; therefore, novel compounds from natural sources, including plants are under exploration. The present study aims to investigate the protective effect of Ocimum sanctum leaf extract (OLE) in CS - induced model of COPD. Exposure to CS was performed thrice a week for 8 weeks and OLE (200 mg/kg and 400 mg/kg) was administered an hour before CS exposure. Control group (negative control) were exposed to ambient air while COPD group was exposed to CS (positive control). Administration of OLE doses reduced inflammation, decreased oxidant concentration and increased antioxidant concentration (p < 0.01). Molecular docking studies between the major phytocompounds of OLE (Eugenol, Cyclohexane and Caryophyllene) and antioxidant enzymes Superoxide dismutase (SOD), Catalase, Glutathione peroxidase (GPx), Glutathione reductase (GR) and Glutathione S Transferase (GST) showed strong binding interaction in terms of binding energy. In vivo and in silico findings for the first time indicates that OLE extract significantly alleviates oxidative stress by its potent free radical scavenging property and strong interaction with antioxidant enzymes. OLE extract may prove to be a therapeutic option for COPD prevention and treatment.

MFE40-the active fraction of Mume Fructus alcohol extract-alleviates Crohn's disease and its complications.

ETHNOPHARMACOLOGICAL RELEVANCE: Mume Fructus (MF) is a well-known traditional Chinese medicine used to treat chronic cough, prolonged diarrhea, and other inflammation-related diseases. We previously confirmed the anti-colitis effect of its ethanol extract on a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced Crohn's disease (CD) rat model. However, the active ingredients and underlying mechanisms of MF remain unknown. AIM OF THE STUDY: To clarify the material basis and potential mechanism of the ethanol extract of MF (MFE) in alleviating CD and its complications, such as lung injury and intestinal obstruction. MATERIALS AND METHODS: MF was extracted with 80% ethanol aqueous solution and separated with 0, 40, and 100% ethanol aqueous solutions. MFE and its fractions were screened in a TNBS-induced CD rat model. For the bioactive fraction, the chemical composition was identified and quantified using ultrahigh-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight tandem mass spectrometry. Interleukin (IL)-1ß, IL-6, IL-17, transforming growth factor (TGF)-ß, and lipopolysaccharide (LPS) levels in the colon, lungs, and/or plasma were detected using enzyme-linked immunosorbent assays. The expression levels of zonula occludens-1 (ZO-1) and occludin in the colon were measured using immunohistochemical staining, and the intestinal microbiota and short-chain fatty acid (SCFA) levels were analyzed using 16S rRNA gene sequencing and gas chromatography/mass spectrometry. RESULTS: The 40% ethanol fraction of MF (MFE40), which mainly contained methyl citrate, ethyl citrate, and caffeoylquinic acid ethyl esters, was identified as the active fraction that could alleviate CD in rats. MFE40 could ameliorate inflammation and fibrosis in the colon and lung tissues by inhibiting the secretion of cytokines, such as IL-1ß, IL-6, IL-17, and TGF-ß, along with intestinal obstruction and lung injury in CD rats. The possible mechanisms of MFE40 were related to increased expression of ZO-1 and occludin in the colon, reduction in plasma LPS levels, and restoration of SCFAs via reduction in the relative abundance of Adlercreutzia, Clostridium_sensu_stricto_1, Erysipelatoclostridium, Faecalibaculum, norank_f_Erysipelotrichaceae, Phascolarctobacterium Coriobacteriaceae_UGG_002, and Allobaculum and increase in the relative abundance of Escherichia shigella, Christensenella, Acetivibrio_ethanolgignens, and Butyricicoccus. MFE40 had no significant influence on the inflammatory factors in healthy rats. CONCLUSIONS: Citrate esters and hydroxycinnamate esters are the main active constituents of MFE40. MFE40 exhibited a remission effect on CD rats and inhibited intestinal obstruction and lung injury via anti-inflammatory effects and regulation of the intestinal microbiota-gut-lung homeostasis.

Nitrated fatty acid, 10-nitrooleate protects against hyperoxia-induced acute lung injury in mice.

The antioxidant and anti-inflammatory effects of electrophilic nitrated fatty acid (NFA); 10-nitrooleate, have been reported. The present study investigated whether 10-nitrooleate has a protective role against hyperoxic-induced acute lung injury (HALI). Using a C57BL/6 mice model of HALI, we investigated the protective effect of 10-nitrooleate. C57BL/6 mice were administered with NFA intratracheally, exposed to hyperoxia for 48 h to induce HALI, and kept at room air for 24 h. Bronchoalveolar lavage (BAL) fluid and lung samples were collected after 24 h of post hyperoxia to analyze markers associated with HALI. Intratracheal (IT) and intraperitoneal (IP) administration of NFA notably attenuated hyperoxia-induced infiltration of inflammatory cells, alveolar-capillary leakage, upregulation of proinflammatory cytokine levels (IL-6 and TNFα) into the BAL fluid, and resolution of inflammation in the lung. Western blot analyses showed that 10-nitrooleate reduced the expression of the inflammatory transcription factor NFκB p65 subunit and increased antioxidant proteins HO-1 and NQO1 expression in the lung tissues compared to vehicle-treated animals. Moreover, 10-nitrooleate reversed the hyperoxia-induced expression of mitophagy-associated markers (PINK1 and p62/SQSTM1), thereby protecting the HALI/ acute respiratory distress syndrome (ARDS). IT and IP delivery of 10-nitrooleate reduces hyperoxia-induced ALI/ARDS by regulating the antioxidant pathways and restoring the mitochondrial homeostasis by regulating mitophagy. It is suggested that NFAs can be further evaluated as supplementary therapy for critically ill patients like COVID-19/ARDS.

Bovine Lactoferrin Alleviates Pulmonary Lipid Peroxidation and Inflammatory Damage in Heat Stroke Rats.

Lung injury occurring in the early stage of heat stroke (HS) leads to hypoxia and further aggravation of other organic damage. Lactoferrin (LF) is an iron binding protein with anti-inflammatory and antioxidant effects. This study focuses on the protection of preadministration of bovine lactoferrin (BLF) against lung injury in rats with HS. Sixty-four Sprague-Dawley male rats were divided into four groups randomly: control (CON)+phosphate-buffered saline (PBS) (n = 16), HS+PBS (n = 16), HS+low-dose BLF (LBLF) (n = 16), and HS+high-dose BLF (HBLF) (n = 16). CON+PBS and HS+PBS were preadministered 10 mL/kg PBS for 1 week. HS+LBLF and HS+HBLF were preadministered 100 and 200 mg/kg BLF for 1 week, respectively. The HS onset time and the survival rate were recorded, and bronchoalveolar lavage fluid was obtained to measure protein concentration. Lung was obtained for pathological analysis and wet/dry weight ratio measurement; later, the content of malondialdehyde (MDA), activity of myeloperoxidase (MPO), and superoxide dismutase (SOD) were measured in lung tissue homogenate. The results indicated that BLF preadministration could delay the HS onset time, enhance the survival rate, the levels of serum inflammatory cytokine and MDA content in HS+LBLF and HS+HBLF showed significant reduction compared with HS+PBS, while a significant elevation of SOD activity and reduction of MPO activity in HS+HBLF. Our results demonstrate that BLF preadministration could relieve lung injury in HS rats by enhancing thermal endurance, and alleviating serum inflammatory response and pulmonary oxidative stress damage.

Selected Phytochemicals to Combat Lungs Injury: Natural Care.

The human has two lungs responsible for respiration and drug metabolism. Severe lung infection caused by bacteria, mycobacteria, viruses, fungi, and parasites may lead to lungs injury. Smoking and tobacco consumption may also produce lungs injury. Inflammatory and pain mediators are secreted by alveolar macrophages. The inflammatory mediators, such as cytokines, interleukin (IL)-1, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α, neutrophils, and fibroblasts are accumulated in the alveoli sac, which becomes infected. It may lead to hypoxia followed by severe pulmonary congestion and the death of the patient. There is an urgent need for the treatment of artificial respiration and ventilation. However, the situation may be the worst for patients suffering from lung cancer, pulmonary tuberculosis, and acute pneumonia caused by acute respiratory distress syndrome (ARDS). Re-urgency has been happening in the case of coronavirus disease of 2019 (COVID-19) patients. Therefore, it is needed to protect the lungs with the intake of natural phytomedicines. In the present review, several selected phyto components having the potential role in lung injury therapy have been discussed. Regular intake of natural vegetables and fruits bearing these constituents may save the lungs even in the dangerous attack of SARS-CoV-2 in lung cancer, pulmonary TB, and pneumatic patients.

Geraniol relieves mycoplasma pneumonia infection-induced lung injury in mice through the regulation of ERK/JNK and NF-κB signaling pathways.

BACKGROUND: Pneumonia is a serious pediatric lung injury disease caused by Mycoplasma pneumoniae (M. pneumoniae) with increasing global prevalence every year. The WHO has reported that nearly 19% of children die due to pneumonia worldwide. OBJECTIVE: The present research was conducted to discover the ameliorative properties of geraniol against M. pneumoniae-provoked pneumonia in mice through the modulation of inflammatory responses. METHODOLOGY: The pneumonia was provoked in the male Swiss albino mice via infecting animals with 100 µl of M. pneumoniae for 2 days and supplemented concurrently with 20 mg/kg of geraniol for 3 days. 100 mg/kg of azithromycin was used as a standard drug. The nitric oxide (NO) level and MPO activity were measured using kits. The SOD activity, GSH, and MDA levels were studied using standard methods. The polymerase chain reaction (PCR) study was performed to examine the M. pneumoniae DNA load. The inflammatory cytokines status was assessed by assay kits. The ERK1/2, JNK1/2, and NF-κB expressions were studied by reverse-transcription (RT-PCR). The lung tissues were analyzed microscopically to investigate the histological alterations. RESULTS: Geraniol treatment effectively reduced lung weight, NO level, and MPO activity in the pneumonia mice. The total cells and M. pneumoniae DNA load were also decreased by the geraniol. The SOD activity and GSH level were improved and MDA was decreased by the geraniol treatment. The IL-1, IL-6, IL-8, TNF-α, and TGF status were appreciably depleted by the geraniol in the pneumonia mice. Geraniol also suppressed the ERK1/2 and NF-κB expressions in the lung tissues. Histological findings also suggest the therapeutic roles of geraniol against pneumonia in mice. CONCLUSION: In summary, our results proved the beneficial roles of geraniol against the M. pneumoniae-provoked pneumonia. Geraniol could be a hopeful therapeutic agent to treat pneumonia in the future.

Lycopene hampers lung injury due to skeletal muscle ischemia-reperfusion in rat model.

This study investigates lycopene's preventive efficacy in skeletal muscle ischemia-reperfusion (I/R) induced lung injury. Thirty-two rats were randomly assigned to control group, lycopene group, I/R group and I/R + lycopene group. In the lycopene and I/R + lycopene groups, the rats initially received 10 mg/kg/day lycopene orally for 15 days. Then, dissection around the abdominal aorta was performed in all rats under general anesthesia. The aorta was clamped at the infrarenal level in the I/R group and I/R + lycopene group for two hours before two hours of reperfusion. The mean serum levels of malondialdehyde (53.0 ± 20.14 nmol/mL) and superoxide dismutase (1.03 ± 0.16 U/mL) were higher and lower in the I/R group than the other three groups, respectively (p < 0.001). The mean serum IMA level of I/R + lycopene group (0.42 ± 0.04 abs/u) was lower than the I/R group (0.47 ± 0.04 abs/u) (p = 0.015). The mean tissue malondialdehyde levels of I/R group (69.10 ± 11.55 nmol/mL) and I/R + lycopene group (68.36 ± 21.17 nmol/mL) were high compared to the control group (49.87 ± 6.52 nmol/mL) and lycopene group (47.82 ± 4.44 nmol/mL) (p = 0.002). The mean tissue glutathione peroxidase (p < 0.001) and superoxide dismutase (p = 0.001) levels of I/R group (121.81 ± 43.59 nmol/mL and 25.17 ± 8.69 U/mL) were low compared to the control group (236.12 ± 18.01 nmol/mL and 46.30 ± 5.17 U/mL), lycopene group (227.52 ± 16.92 nmol/mL and 45.82 ± 4.02 U/mL), and I/R + lycopene group (176.02 ± 24.27 nmol/mL and 35.20 ± 4.85 U/mL). The histopathological analyses of I/R + lycopene group indicated less significant changes than the control group. Tissue damage in the I/R + lycopene group was less prominent than the I/R group. These findings suggest oral lycopene supplementation as a promising prevention against skeletal muscle I/R caused lung injury.

Selenomethionine alleviates LPS-induced JNK/NLRP3 inflammasome-dependent necroptosis by modulating miR-15a and oxidative stress in chicken lungs.

Selenium (Se) was involved in many physiological processes in humans and animals. microRNAs (miRNAs) also played important roles in lung diseases. However, the regulatory mechanism of miRNA in chicken lungs and the mechanism of lipopolysaccharide (LPS)-induced pneumonia remained unclear. To further study these mechanisms, we established a supplement of selenomethionine (SeMet) and/or LPS-treated chicken model and a cell model of LPS and/or high and low expression of miR-15a in chicken hepatocellular carcinoma (LMH) cells. We detected the expression of some selenoproteins, p-c-Jun N-terminal kinase (JNK), nod-like receptor protein 3 (NLRP3), caspase1, receptor-interacting serine-threonine kinase 1 (RIPK1), receptor-interacting serine-threonine kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL), miR-15a, and oxidative stress kits. Additionally, we observed the morphology of lungs by H.E. staining in vitro. The results indicated that necroptosis occurred in LPS-treated chicken and LMH cells. Moreover, LPS stimulation inhibited miR-15a, and increased the expression of JNK, NLRP3, caspase1, RIPK1, RIPK3, and MLKL. We also found that LPS treatment not only increased the content of H2O2 and MDA in the lungs but also increased the activities of iNOS and CAT and the content of GSH decreased. Conclusion: SeMet could reduce the oxidative damage and activate NLRP3 inflammasome reaction by stimulating miR-15a/JNK, thus reduced the pulmonary necroptosis induced by LPS.

Eurotium cristatum Fermented Loose Dark Tea Ameliorates Cigarette Smoke-Induced Lung Injury by MAPK Pathway and Enhances Hepatic Metabolic Detoxification by PXR/AhR Pathway in Mice.

Cigarette smoke- (CS-) induced oxidative stress and inflammation in the lung are serious health problems. Primary and reprocessed tea products contain multiple antioxidants that have been reported to protect the lung against CS-induced injury. However, the beneficial effects of Eurotium cristatum fermented loose dark tea (ECT) and Eurotium cristatum particle metabolites (ECP) on CS-induced lung injury and its potential hepatic metabolic detoxification are still unclear. Therefore, sixty mice were randomly divided into six equal groups. CS-exposed mice were prevented or treated with ECP or ECT infusions for 12 or 8 weeks to determine the antioxidative stress, anti-inflammatory and potential metabolic detoxification of ECT and ECP. Thirty-six mice were randomly divided into six equal groups to observe the effects on hepatic metabolic detoxification by replacing daily drinking water with ECT. Results showed that CS significantly decreased the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and upregulated the expressions of malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, and IL-1ß in serum. These adverse effects were modulated by ECP and ECT. In addition, ECT upregulated the mRNA expression of pregnane X receptor (PXR) and cytochrome P450 (CYP450) in the liver on daily free drinking ECT mice group. Western blot analysis further revealed that in CS-exposed mice, ECP and ECT significantly decreased the phosphorylation of mitogen-activated protein kinase (MAPK) in the lung but upregulated the protein expressions of PXR and aryl hydrocarbon receptor (AhR) in the liver. Overall, our findings demonstrated that ECT and ECP protected against lung injury induced by CS via MAPK pathway and enhanced hepatic metabolic detoxification via PXR and AhR pathways. Therefore, daily intake of ECT and ECP can potentially protect against CS-induced oxidative and inflammatory injuries.

Tea polyphenols and Levofloxacin alleviate the lung injury of hepatopulmonary syndrome in common bile duct ligation rats through Endotoxin -TNF signaling.

BACKGROUND & AIMS: Hepatopulmonary syndrome (HPS) is characterized by pulmonary vasodilation and arterial blood oxygen desaturation in patients with chronic liver disease. Generally, common bile duct ligation (CBDL) rats are a suitable experimental model for studying hepatopulmonary syndrome. Our previous study demonstrated that endotoxin surges markedly, followed by bacterial translocation and the loss of liver immune function in all the stages of CBDL, thereby contributing to the pathogenesis of HPS. However, the mechanisms behind the increase of the endotoxin and how to alleviate it have not yet been elucidated. Pulmonary injury induced by increased bilirubin, endotoxin, and inflammatory mediators occurs in the early and later stages of CBDL. This study assessed the effects of Tea polyphenols (TP) and Levofloxacin on endotoxin reduction and suppression of lung injury in HPS rats in the long and short term, respectively. METHODS: Morphological change of pulmonary injury, HPS relative index, endotoxin concentration, and the activation extent of Malondialdehyde (MDA) and Myeloperoxidase (MPO) were evaluated in CBDL rats with or without TP and Levofloxacin for three weeks or six weeks. The inflammation factors of serum, lung tissue, and BALF were then compared at the same condition for the two time periods. This was followed by adoption of the network pharmacology approach, which was mainly composed of active component gathering, target prediction, HPS gene collection, network analysis, and gene enrichment analysis. Finally, the mRNA and protein levels of the inflammatory factors were studied and relative signaling expression was assessed using RT-PCR and Western blot analysis. RESULTS: The obtained results indicated that the pulmonary injury manifestation was perceived and endotoxin, MDA, and MPO activation were markedly increased in the early and later stages of CBDL. TP and Levofloxacin treatment alleviated endotoxin infection and inflammation factor expression three weeks and six weeks after CBDL. In addition, Levofloxacin displayed a short time anti-bacterial effect, while TP exerted a long period function. TP and Levofloxacin also reduced TNF-α, TGF-ß, IL-1ß, PDGF-BB, NO, ICAM-1, and ET-1 expression on the mRNA or protein expression. With regard to the pharmacological mechanism, the network analysis indicated that 12 targets might be the therapeutic targets of TP and Levofloxacin on HPS, namely ET-1, NOs3, VEGFa, CCl2, TNF, Ptgs2, Hmox1, Alb, Ace, Cav1, and Mmp9. The gene enrichment analysis implied that TP and Levofloxacin probably benefited patients with HPS by modulating pathways associated with the AGE-RAGE signaling pathway, the TNF signaling pathway, the HIF-1 signaling pathway, the VEGF signaling pathway, and the IL-17 signaling pathway, Rheumatoid arthritis, Fluid shear stress, and atherosclerosis. Finally, the TNF-α level was mainly diminished on the protein level following CBDL. CONCLUSIONS: TP and Levofloxacin could alleviate pulmonary injury for short and long period, respectively, while at the same time preventing endotoxin and the development of HPS in CBDL rats. These effects are possibly associated with the regulation of the Endotoxin -TNF-α pathways.

Vitex negundo Linn. extract alleviates inflammatory aggravation and lung injury by modulating AMPK/PI3K/Akt/p38-NF-κB and TGF-ß/Smad/Bcl2/caspase/LC3 cascade and macrophages activation in murine model of OVA-LPS induced allergic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: There is growing inclination towards developing bioactive molecule-based strategies for the management of allergic airway inflammation associated respiratory diseases. Vitex negundo Linn., also known as Nirgundi, is one such medicinal plant enriched with phytochemicals and used for inflammatory and respiratory disorders including asthma in traditional system of medicine. Preliminary studies have claimed anti-tussive and bronchodilator potential of V. negundo Linn. However, its attributes as well as molecular mechanism (s) in modulation of asthma mediated by allergic inflammation are yet to be delineated scientifically. AIM OF THE STUDY: Present study attempted to assess the effectiveness of Vitex negundo leaf extract (VNLE) in mitigation of allergen induced inflammation associated asthmatic lung damage with emphasis to delineate its molecular mechanism (s). MATERIALS AND METHODS: Allergic lung inflammation was established in Balb/c mice using Ovalbumin-lipopolysaccharide (OVA-LPS). Several allergic inflammatory parameters, histopathological changes, alveolar macrophage activation and signalling pathways were assessed to examine protective effects of VNLE. UHPLC-DAD-QTOF-ESI-IMS was used to characterize VLNE. RESULTS: VNLE administration effectively attenuated LPS-induced oxi-inflammatory stress in macrophages suggesting its anti-inflammatory potential. Further, VNLE showed protective effect in mitigating asthmatic lung damage as evident by reversal of pathological changes including inflammatory cell influx, congestion, fibrosis, bronchial thickness and alveolar collapse observed in allergen group. VNLE suppressed expressions of inflammatory Th1/Th2 cytokines, chemokines, endopeptidases (MMPs), oxidative effector enzyme (iNOS), adhesion molecules, IL-4/IFN-γ release with simultaneous enhancement in levels of IL-10, IFN-γ, MUC3 and tight junction proteins. Subsequent mechanistic investigation revealed that OVA-LPS concomitantly enhanced phosphorylation of NF-κB, PI3K, Akt and p38MAPKs and downregulated AMPK which was categorically counteracted by VNLE treatment. VNLE also suppressed OVA-LPS induced fibrosis, apoptosis, autophagy and gap junction proteins which were affirmed by reduction in TGF-ß, Smad2/3/4, Caspase9/3, Bax, LC3A/B, connexin 50, connexin 43 and enhancement in Bcl2 expression. Additionally, suppression of alveolar macrophage activation, inflammatory cells in blood and elevation of splenic CD8+T cells was demonstrated. UHPLC-DAD-QTOF-ESI-IMS revealed presence of iridoids glycoside and phenolics which might contribute these findings. CONCLUSION: These findings confer protective effect of VNLE in attenuation of allergic lung inflammation and suggest that it could be considered as valuable medicinal source for developing safe natural therapeutics for mitigation of allergic inflammation during asthma.

Astaxanthin Prevents Lung Injury Due to Hyperoxia and Inflammation.

BACKGROUND/AIM: This study aimed to ascertain the effects of astaxanthin on the lungs of rat pups with bronchopulmonary dysplasia (BPD) induced by hyperoxia and lipopolysaccharide (LPS). MATERIALS AND METHODS: Forty-two newborn Wistar rats, born to spontaneous pregnant rats, were divided into three groups: Hyperoxia (95% O2) + lipopolysaccharide (LPS) group, hyperoxia + LPS + astaxhantin group, and control: no treatment group (21% O2). Pups in the hyperoxia + LPS + astaxanthin group were given 100 mg/kg/day oral astaxanthin from the first day to the fifth day. Histopathologic and biochemical evaluations, including glutathione (GSH), total anti-oxidant status (TAS), total oxidant status (TOS), lipid hydroperoxide (LPO), 8-hydroxydeoxyguanosine (8-OHdG), advanced oxidation protein products (AOPP), myeloperoxidase (MPO), total thiol, tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1ß), and caspase-3 activities, were performed. RESULTS: Better survival rates and weight gain were demonstrated in the hyperoxia + LPS + astaxanthin group (p <0.001). In the histopathologic evaluation, the severity of lung damage was significantly reduced in the hyperoxia+LPS+astaxanthin group, as well as decreased apoptosis (ELISA for caspase-3) (p <0.001). The biochemical analyses of lung tissues showed that TAS, GSH, and Total thiol levels were significantly higher in the astaxanthin treated group compared to the hyperoxia + LPS group (p <0.05) while TOS, AOPP, LPO, 8-OHdG, MPO levels were significantly lower (p <0.001). In addition, unlike the hyperoxia + LPS group, TNF-α and IL-1ß levels in lung tissue were significantly lower in the astaxanthin-treated group (p <0.001). CONCLUSION: Astaxanthin was shown to reduce lung damage caused by inflammation and hyperoxia with its anti-inflammatory, anti-oxidant, anti-apoptotic properties, and to protect the lung from severe destruction.

Gut microbiota modulation and anti-inflammatory properties of Xuanbai Chengqi decoction in septic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanbai Chengqi decoction (XBCQ), a traditional Chinese medicine formulation, was reported to have a protective role in a variety of pulmonary infection diseases. However, its mechanism remains uncertain. In the current study, we investigated the potential mechanism of XBCQ, its therapeutic effects on organ injuries induced by sepsis and gut microbiota modulation. MATERIAL AND METHODS: 80 Male Sprague Dawley rats were performed cecal ligation and puncture (CLP) for sepsis model and 60 of them were treated with different doses of XBCQ (3.78, 7.56, 15.12 g/Kg, 20 rats per group) twice per day. After the most valid dose was determined, another 40 rats were divided randomly into four groups: sham group, sham + XBCQ group, sepsis group, sepsis + XBCQ group. The sepsis + XBCQ group was treated with XBCQ by intragastric administration and then twice per day. Feces of the rats were collected and the gut microbiota constituents were analyzed by 16S rDNA sequencing. Histological changes were observed by H&E staining. Occludin content in the colon was determined by immunohistochemical analysis. The concentrations of cytokines were determined by enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: The survival rate of septic rats was increased significantly at the dose of 7.56 g/Kg from 50% to 80% at 72 h. The gut microbiota richness and composition were disturbed in septic rats. XBCQ altered the gut microbiota, involving alpha diversity changes, significantly reducing the relative abundance of Bacteroidaceae and ClostridiumXI and increasing that of Firmicutes and Actinobacteria. Furthermore, the relative abundances of Lactobacillus, Butyricicoccus and Bifidobacterium were increased by XBCQ. Moreover, the gut barrier dysfunction was improved by XBCQ through restoring the impaired tight conjunction protein Occludin. The concentration of diamine oxidase was decreased, while the D-lactate level was elevated. Meanwhile, the level of myeloperoxidase (MPO) in the lung tissue of the XBCQ-treated group was reduced. Lung injury was also alleviated by decreased levels of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß) and interleukin 10 (IL-10) in bronchoalveolar lavage fluids (BALFs). The relative abundance of potential microbial biomarkers in four groups significantly correlated with the concentration of inflammatory factors in BALFs. CONCLUSIONS: Our results suggested that XBCQ had a protective role against sepsis by modulating the gut microbiota, restoring the intestinal epithelial barrier and decreasing inflammatory responses.

Establishment of an in vitro model using the rat alveolar macrophage cell line NR8383.

OBJECTIVE: To establish an in vitro model of radiation-induced lung injury using rat lung alveolar macrophages (NR8383). METHODS: Using a medical electronic linear accelerator, cells were irradiated with either 0 Gy or 6 Gy X-rays. At 6, 12, 24, 30 and 48 h, the DNA damage index (8-OHdG) and lipid damage index (MDA) were measured in the two groups. We also determined the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and transforming growth factor-ß (TGF-ß). RESULTS: The levels of 8-OHdG and MDA in the 6 Gy irradiation group were higher than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. The levels reached the highest value -6 h after irradiation, and then gradually decreased. The levels of the inflammatory factors TNF-α, TGF-ß and IL-6 were higher in the 6 Gy irradiation group than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. CONCLUSION: Six Gy X-ray irradiated NR8383 cells can be used to establish an in-vitro model of radiation-induced lung injury. The levels of 8-OHdG, MDA, TNF-α, TGF-ß and IL-6 can be used as effective evaluation indicators.

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.