Histological Changes In Lungs Research Articles

Sangju Cold Granule exerts anti-viral and anti-inflammatory activities against influenza A virus in vitro and in vivo

Ethnopharmacological relevanceSangju Cold Granule (SJCG) is a classical traditional Chinese medicine (TCM) prescription described in “Item Differentiation of Warm Febrile Diseases”. Historically, SJCG was employed to treat respiratory illnesses. Despite its popular usage, the alleviating effect of SJCG on influenza A virus infection and its mechanisms have not been fully elucidated. Aim of the studyInfluenza is a severe respiratory disease that threatens human health. This study aims to assess the therapeutic potential of SJCG and the possible molecular mechanism underlying its activity against influenza A virus in vitro and in vivo. Materials and methodsUltrahigh-performance liquid chromatography (UPLC)-Q-Exactive was used to identify the components of SJCG. The 50% cytotoxic concentration of SJCG in MDCK and A549 cells were determined using the CCK-8 assay. The activity of SJCG against influenza A virus H1N1 was evaluated in vitro using cytopathic effect inhibition and progeny virus titer reduction assays. RT-qPCR was performed to obtain the expression levels of inflammatory mediators and the transcriptional regulation of RIG-I and MDA5 in H1N1-infected A549 cells. Then, the mechanism of SJCG effect on viral replication and inflammation was further explored by measuring the expressions of proteins of the RIG-I/NF-kB/IFN(I/III) signaling pathway by western blot. The impact of SJCG was explored in vivo in an intranasally H1N1-infected BALB/c mouse pneumonia model treated with varying doses of SJCG. The protective role of SJCG in this model was evaluated by survival, body weight monitoring, lung viral titers, lung index, lung histological changes, lung inflammatory mediators, and peripheral blood leukocyte count. ResultsThe main SJCG chemical constituents were flavonoids, carbohydrates and glycosides, amino acids, peptides, and derivatives, organic acids and derivatives, alkaloids, fatty acyls, and terpenes. The CC50 of SJCG were 24.43 mg/mL on MDCK cells and 20.54 mg/mL on A549 cells, respectively. In vitro, SJCG significantly inhibited H1N1 replication and reduced the production of TNF-α, IFN-β, IL-6, IL-8, IL-13, IP-10, RANTES, TRAIL, and SOCS1 in infected A549 cells. Intracellularly, SJCG reduced the expression of RIG-I, MDA5, P-NF-κB P65 (P-P65), P-IκBα, P-STAT1, P-STAT2, and IRF9. In vivo, SJCG enhanced the survival rate and decreased body weight loss in H1N1-infected mice. Mice with H1N1-induced pneumonia treated with SJCG showed a lower lung viral load and lung index than untreated mice. SJCG effectively alleviated lung damage and reduced the levels of TNF-α, IFN-β, IL-6, IP-10, RANTES, and SOCS1 in lung tissue. Moreover, SJCG significantly ameliorated H1N1-induced leukocyte changes in peripheral blood. ConclusionsSJCG significantly reduced influenza A virus and virus-mediated inflammation through inhibiting the RIG-I/NF-kB/IFN(I/III) signaling pathway. Thus, SJCG could provide an effective TCM for influenza treatment.

The Prostaglandin D2 Receptor CRTH2 Contributes to Airway Hyperresponsiveness during Airway Inflammation Induced by Sensitization without an Adjuvant in Mice.

Prostaglandin D2 (PGD2), which is produced mainly by Th2 cells and mast cells, promotes a type-2 immune response by activating Th2 cells, mast cells, eosinophils, and group 2 innate lymphoid cells (ILC2s) via its receptor, chemoattractant receptor-homologous molecules on Th2 cells (CRTH2). However, the role of CRTH2 in models of airway inflammation induced by sensitization without adjuvants, in which both IgE and mast cells may play major roles, remain unclear. Wild-type (WT) and CRTH2-knockout (KO) mice were sensitized with ovalbumin (OVA) without an adjuvant and then challenged intranasally with OVA. Airway inflammation was assessed based on airway hyperresponsiveness (AHR), lung histology, number of leukocytes, and levels of type-2 cytokines in the bronchoalveolar lavage fluid (BALF). AHR was significantly reduced after OVA challenge in CRTH2 KO mice compared to WT mice. The number of eosinophils, levels of type-2 cytokines (IL-4, IL-5, and IL-13) in BALF, and IgE concentration in serum were decreased in CRTH2 KO mice compared to WT mice. However, lung histological changes were comparable between WT and CRTH2 KO mice. CRTH2 is responsible for the development of asthma responses in a mouse model of airway inflammation that features prominent involvement of both IgE and mast cells.

Lung molecular and histological changes in type 2 diabetic rats and its improvement by high-intensity interval training

BackgroundType 2 diabetes (T2D) leads to serious respiratory problems. This study investigated the effectiveness of high-intensity interval training (HIIT) on T2D-induced lung injuries at histopathological and molecular levels.MethodsForty-eight male Wistar rats were randomly allocated into control (CTL), Diabetes (Db), exercise (Ex), and Diabetes + exercise (Db + Ex) groups. T2D was induced by a high-fat diet plus (35 mg/kg) of streptozotocin (STZ) administration. Rats in Ex and Db + Ex performed HIIT for eight weeks. Tumor necrosis factor-alpha (TNFα), Interleukin 10 (IL-10), BAX, Bcl2, Lecithin, Sphingomyelin (SPM) and Surfactant protein D (SPD) levels were measured in the bronchoalveolar lavage fluid (BALF) and malondialdehyde (MDA) and total antioxidant capacity (TAC) levels were measured in lung tissue. Lung histopathological alterations were assessed by using H&E and trichrome mason staining.ResultsDiabetes was significantly associated with imbalance in pro/anti-inflammatory, pro/anti-apoptosis and redox systems, and reduced the SPD, lecithin sphingomyelin and alveolar number. Performing HIIT by diabetic animals increased Bcl2 (P < 0.05) and IL10 (P < 0.01) levels as well as surfactants components and TAC (P < 0.05) but decreased fasting blood glucose (P < 0.001), TNFα (P < 0.05), BAX (P < 0.05) and BAX/Bcl2 (P < 0.001) levels as well as MDA (P < 0.01) and MDA/TAC (P < 0.01) compared to the diabetic group. Furthermore, lung injury and fibrosis scores were increased by T2D and recovered in presence of HIIT.ConclusionThese findings suggested that the attenuating effect of HIIT on diabetic lung injury mediated by reducing blood sugar, inflammation, oxidative stress, and apoptosis as well as improving pulmonary surfactants components.Graphical Type 2 diabetes increased inflammation, oxidative stress and apoptosis and reduced pulmonary surfactants , while high intensity training improved these negative effects

Roles of Nrf2/HO-1 and ICAM-1 in the Protective Effect of Nano-Curcumin against Copper-Induced Lung Injury.

Copper (Cu) is an essential trace element for maintaining normal homeostasis in living organisms. Yet, an elevated level of Cu beyond homeostatic capacity may lead to oxidative damage of cellular components in several organs, including the lungs. This work investigated the effects of curcumin (Curc) and nano-curcumin (nCurc) against Cu-induced lung injury, accenting the roles of oxidative stress, inflammation, and the nuclear factor erythroid 2-related factor/heme oxygenase-1 Nrf2/HO-1 pathway. Rats were challenged with 100 mg/kg of copper sulfate (CuSO4) while being treated with Curc or nCurc for 7 days. Cu-triggered lung oxidative stress detected as dysregulation of oxidative/antioxidant markers, a downregulation of Nrf-2/HO-1 signaling, and an increase in the inflammatory markers interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and intracellular adhesion molecule-1 (ICAM-1). Additionally, it decreased the expression of lung-specific proteins, surfactant protein-C (SP-C), and mucin-1 (MUC-1), induced apoptosis, and caused changes in lung histology. Curc and nCurc alleviated CuSO4-induced lung injury by suppressing oxidative damage and inflammation and activating Nrf-2/HO-1. They also prevented apoptosis and restored the normal expression of SP-C and MUC-1. We concluded that nCurc exhibited superior efficacy compared with Curc in mitigating CuSO4-induced lung injury. This was associated with reduced oxidative stress, inflammation, and apoptotic responses and increased Nrf2/HO-1 signaling and expression of SP-C and MUC-1.

Arsenic trioxide demonstrates efficacy in a mouse model of preclinical systemic sclerosis

BackgroundUncontrolled T-cell activation plays a key role in systemic sclerosis (SSc). Arsenic trioxide (ATO) has immunological effects and has demonstrated potential in preclinical SSc models. In this study, we assessed the efficacy of ATO in Fra2 transgenic (Fra2TG) mice, which develop severe vascular remodeling of pulmonary arterioles and nonspecific interstitial pneumonia-like lung disease, closely resembling human SSc-associated pulmonary hypertension, therefore partially resembling to the SSc human disease.MethodsThe efficacy of ATO in Fra2TG mice was evaluated through histological scoring and determination of cell infiltration. Fibrotic changes in the lungs were assessed by measuring collagen content biochemically, using second harmonic generation to measure fibrillar collagen, and imaging via computed tomography. Cardiovascular effects were determined by measuring right ventricular systolic pressure and vessel remodeling. The mechanism of action of ATO was then investigated by analyzing lung cell infiltrates using flow cytometry and bulk RNA with sequencing techniques.ResultsAfter ATO treatment, the Ashcroft histological score was substantially decreased by 33% in ATO-treated mice compared to control mice. Other investigations of fibrotic markers showed a trend of reduction in various measurements of fibrosis, but the differences did not reach significance. Further cardiovascular investigations revealed convergent findings supporting a beneficial effect of ATO, with reduced right ventricular systolic pressure and medial wall thickness, and a significant decrease in the number of muscularized distal pulmonary arteries in ATO-treated Fra2TG mice compared to untreated Fra2TG mice. Additionally, inflammatory cell infiltration was also markedly reduced in lesioned lungs. A reduction in the frequency of CD4 + and T effector memory cells, and an increase in the percentage of CD4 + T naive cells in the lungs of ATO-treated Fra-2TG mice, was observed when compared to PBS group Fra-2Tg mice. RNA-seq analysis of ATO-treated mouse lungs revealed a downregulation of biological pathways associated with immune activity and inflammation, such as T-cell activation, regulation of leucocyte activation, leucocyte cell–cell adhesion, and regulation of lymphocyte activation.ConclusionsOur results suggest the clinical relevance of ATO treatment in SSc. Using the Fra2TG mouse model, we observed significant lung histological changes, a trend towards a decrease in various fibrotic makers, and a strong reduction in vascular remodeling. The mechanism of action of ATO appears to involve a marked counteraction of the immune activation characteristic of SSc, particularly T-cell involvement. These findings pave the way for further studies in SSc.

Characterization of Lung Inflammatory Response to Aspergillus fumigatus Spores.

The airway exposure to Aspergillus fumigatus spores (AFsp) is associated with an inflammatory response, potentially leading to allergic and/or chronic pulmonary aspergillosis. The aim of our study is to better understand the host response, first in vitro, then in vivo, following the chronic exposure of mice to AFsp. We investigated the inflammatory response to AFsp in cell mono- and co-culture systems with murine macrophages and alveolar epithelial cells. The mice were subjected to two intranasal instillations using 105 AFsp. Their lungs were processed for inflammatory and histopathological analyses. In cell culture, the gene expressions significantly increased for TNF-α, CXCL-1, CXCL-2, IL-1β, IL-1α and GM-CSF in macrophages, with these increases being limited for TNF-α, CXCL-1 and IL-1α in epithelial cells. In co-culture, increases in the TNF-α, CXCL-2 and CXCL-1 gene expressions were observed to be associated with increased protein levels. The in vivo lung histological analyses of mice challenged by AFsp showed cellular infiltrates in the peribronchial and/or alveolar spaces. A Bio-Plex approach on the bronchoalveolar lavage revealed significant increases in the protein secretion of selected mediators of the challenged mice compared to the unchallenged mice. In conclusion, the exposure to AFsp resulted in a marked inflammatory response of macrophages and epithelial cells. These inflammatory findings were confirmed in mouse models associated with lung histologic changes.

In Vitro and In Vivo Therapeutic Potential of 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea on Cells and K18-hACE2 Mice Infected with SARS-CoV-2.

We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6'-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-κB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and/or its molecular derivatives against this and other related viruses.

Repurposing of carvedilol to alleviate bleomycin-induced lung fibrosis in rats: Repressing of TGF-β1/α-SMA/Smad2/3 and STAT3 gene expressions

Idiopathic pulmonary fibrosis (IPF) is the most widely studied interstitial lung disease. IPF eventually leads to respiratory insufficiency, lung cancer, and death. Carvedilol (CAR) is a third-generation β-adrenergic receptor antagonist with an α1-blocking effect. CAR demonstrates antifibrotic activities in various experimental models of organ fibrosis. Aims: This work is designed to explore the possible alleviating effects of CAR on bleomycin (BLM)-induced lung fibrosis in rats. Main methods: The BLM rat model of lung fibrosis was achieved by intratracheal delivery of a single dose of 5 mg/kg of BLM. Seven days following BLM injection, either prednisolone or CAR was orally administered at doses of 10 mg/kg once daily for 21 days to the rats. The actions of CAR were evaluated by lung oxidant/antioxidant parameters, protein concentration and total leucocyte count (TLC) in bronchoalveolar lavage fluid (BALF), fibrosis regulator-related genes along with the coexistent lung histological changes. Key findings: CAR effectively decreased lung malondialdehyde level, increased superoxide dismutase activity, declined both protein concentration and TLC in BALF, downregulated TGF-β1/α-SMA/Smad2/3 and STAT3 gene expressions, and repaired the damaged lung tissues. Significance: CAR conferred therapeutic potential against BLM-induced lung fibrosis in rats, at least in part, to its antioxidant, anti-inflammatory, and antifibrotic activities. CAR could be utilized as a prospective therapeutic option in patients with lung fibrosis in clinical practice.

Emodin alleviates lung ischemia-reperfusion injury by suppressing gasdermin D-mediated pyroptosis in rats.

Pyroptosis refers to programmed cell death associated with inflammation. Emodin has been reported to alleviate lung injuries caused by various pathological processes and attenuate ischemia-reperfusion (I/R) injuries in diverse tissues. Lewis rats were assigned into the sham, the I/R, and the I/R + emodin groups. Emodin and phosphate-buffered saline were intraperitoneally injected into rats of the emodin group and I/R group for 30 min, respectively. These rats were then subjected to left thoracotomy followed by 90-min clamping of the left hilum and 120-min reperfusion. Sham-operated rats underwent 210-min ventilation. Lung functions, histological changes, lung edema, and cytokine levels were assessed. Protein levels were measured by western blotting. Immunofluorescence staining was conducted to evaluate pyroptosis. Emodin alleviated the I/R-induced lung dysfunction, lung damages, and inflammation. Protective effects of emodin against I/R-mediated endothelial pyroptosis was observed in vivo and in vitro. Mechanistically, emodin inactivated the TLR4/MyD88/NF-κB/NLRP3 pathway. Emodin attenuates lung ischemia-reperfusion injury by inhibiting GSDMD-mediated pyroptosis in rats.

Nicotinamide Mononucleotide Ameliorates Silica-Induced Lung Injury through the Nrf2-Regulated Glutathione Metabolism Pathway in Mice

Nicotinamide mononucleotide (NMN) is a natural antioxidant approved as a nutritional supplement and food ingredient, but its protective role in silicosis characterized by oxidative damage remains unknown. In this study, we generated a silicosis model by intratracheal instillation of silica, and then performed histopathological, biochemical, and transcriptomic analysis to evaluate the role of NMN in silicosis. We found that NMN mitigated lung damage at 7 and 28 days, manifested as a decreasing coefficient of lung weight and histological changes, and alleviated oxidative damage by reducing levels of reactive oxygen species and increasing glutathione. Meanwhile, NMN treatment also reduced the recruitment of inflammatory cells and inflammatory infiltration in lung tissue. Transcriptomic analysis showed that NMN treatment mainly regulated immune response and glutathione metabolism pathways. Additionally, NMN upregulated the expression of antioxidant genes Gstm1, Gstm2, and Mgst1 by promoting the expression and nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2). Gene interaction analysis showed that Nrf2 interacted with Gstm1 and Mgst1 through Gtsm2. Promisingly, oxidative damage mediated by these genes occurred mainly in fibroblasts. In summary, NMN alleviates silica-induced oxidative stress and lung injury by regulating the endogenous glutathione metabolism pathways. This study reveals that NMN supplementation might be a promising strategy for mitigating oxidative stress and inflammation in silicosis.

Abstract 15507: The Role of Obesity in Covid-19 Pathogenesis and Inflammation

SARS-CoV-2 has caused a global pandemic unseen for nearly a century. To date hundreds of millions of people have been diagnosed and millions more have died from COVID-19 disease; many of which are in groups with comorbidities. Obesity is associated with a 3.07 times higher risk of hospitalization and a 1.42 times higher risk of severe illness. However, our understanding of obesity-accelerated severity of COVID-19 is limited. To model the increased disease severity of obese individuals with COVID-19, we used a humanized ACE2 (hACE2) transgenic mouse model, in which hACE2 is under the cytokeratin-18 promoter (K18). Modeling the effects of obesity on COVID-19 in K18-hACE2 mice will help us better understand the mechanisms underlying obesity-accelerated severity of COVID-19 disease and inflammation. We have developed a study in which K18 hACE2 mice infected with SARS-CoV-2 were fed both a normal standard diet as well as a western high fat and carbohydrate diet that causes them to gain excess weight and become obese over a period of 3 months. K18-hACE2 obese mice lose a signifyingly more percentage body weight over both a 7 (acute) 14 day (long-term) period than normal diet fed mice do after infection with SARS-CoV-2. H&amp;E staining analysis of lung histological changes indicated a dramatic increase in inflammation in our K18 hACE2 obese mice infected with SARS-CoV-2 . Subgenomic qPCR and IHC staining was used to quantify virus (viral titer) which indicated that in both of our diet groups after infection did not have a significantly different SARS-CoV-2 viral load. No significant difference in the SARS-CoV-2 viral load was detected at either 7 or 14 dpi with SARS-CoV-2 . We further measured adipokines with an ELISA on two of the most notable adipokines: leptin and adiponectin. Levels of leptin were significantly higher in our obese K18-hACE2 mice but not in our normal mice after infection. There was no difference however in the levels of adiponectin. Obese K18-hACE2 mice show a more severe COVID 19 phenotype than normal diet mice after infection and can be used to further study the underlying mechanism of how SARS-CoV-2 infection in obese individuals causes a more severe disease phenotype.

Abstract 13194: Towards Understanding the Pathogenesis of Covid-19: Acute and Long-Term Mouse Models

SARS-CoV2 (CoV2) infection causes both acute and long-term health effects via damaging multiple organs including lung. The endothelial dysfunction associated with the infection may contribute to pathogenesis of acute COVID-19 and long COVID. However, the mechanisms underlying the endothelial dysfunction remain elusive. Development of mouse models for these diseases will help us better dissect these mechanisms and facilitate the development of therapeutics for treatment of the disease. Previously, we developed an acute COVID model by infecting human ACE2 transgenic (K18) mice with a lethal CoV2 dose. K18 mice developed severe COVID-19, including progressive body weight loss and fatality at days 7 post infection (DPI), severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. We detected CoV2 in capillary endothelial cells, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs in this model. These results indicate that CoV2 infection and infection-mediated immune activation caused endothelial dysfunction, which contributes to the pathogenesis of severe COVID-19. To further develop a model for long COVID, we infected K18 mice with sub lethal CoV2 dose, monitored the body weight and survival rate and characterized the lung and brain histological changes at 21 and 45 DPI. The infected mice progressively lost body weights from 5 to 7 DPI and started to rebound from 8 DPI and then returned to baseline at 13 DPI. Mice had extensive patchy inflammation in the lungs associated with collagen deposition and smooth muscle action expression. We also found moderate levels of total viral RNA in the lung but not brain while viral subgenomic RNA (a correlate of viral replication) was undetectable in lung or brain by qRT-PCR assay. Fluorescence staining showed co-localization of CoV2 spike protein and CD206 in lungs, suggesting macrophage engulfment CoV2 at late time points. Together, we have successfully established long-term COVID mouse models, which will be useful tools for further defining the role of endothelial dysfunction in pathogeneses of CoV2-related acute and long COVID.

Adipose tissue-derived mesenchymal stem cells attenuate lung inflammation and fibrosis in the bleomycin-induced pulmonary fibrosis rat model via caveolin-1/NF-kB signaling axis.

Stem cells have emerged as promising therapeutic options for several human diseases, including pulmonary fibrosis (PF). In this study, we investigated the therapeutic effects of adipose tissue-derived mesenchymal stem cells (ADMSCs) in the bleomycin-induced PF model rats and the underlying mechanisms. The PF model rats were generated by intratracheal injections of 5 mg/kg bleomycin sulfate. The ADMSC group rats were generated by injecting 2×10(6) ADMSCs via the tail vein at 0, 12, and 24 h after bleomycin injection. The control, PF, and ADMSC group rats were sacrificed on day 21 after bleomycin injections and the changes in lung histology and the levels of pro-inflammatory cytokines, collagen I, and caveolin-1 (Cav-1), and the activity of the NF-kappaB signaling pathway in the lung tissues was assessed by hematoxylin-eosin staining, ELISA, and western blotting assays. The lung tissues of the PF model rats showed significant infiltration of neutrophils, tissue destruction, and collagen deposition, but these effects were abrogated by the ADMSCs. The levels of pro-inflammatory cytokines such as IL-6, IL-1beta, and TGF-beta1 were elevated in the lung tissues and the bronchoalveolar lavage fluid (BALF) of the bleomycin-induced PF model rats, but these effects were reversed by the ADMSCs. The lung tissues of the PF model rats showed significant downregulation of Cav-1 and significantly higher activation of the pro-inflammatory NF-kappaB pathway. However, administration of the ADMSCs restored the expression levels of Cav-1 and suppressed the NF-kappaB signaling pathway in the lungs of the bleomycin-induced PF model rats. In conclusion, this study demonstrated that the ADMSCs protected against bleomycin-induced PF in the rat model by modulating the Cav-1/NF-kappaB axis.

5-Methoxyflavone-induced AMPKα activation inhibits NF-κB and P38 MAPK signaling to attenuate influenza A virus-mediated inflammation and lung injury in vitro and in vivo

Influenza-related acute lung injury (ALI) is a life-threatening condition that results mostly from uncontrolled replication of influenza virus (IV) and severe proinflammatory responses. The methoxy flavonoid compound 5-methoxyflavone (5-MF) is believed to have superior biological activity in the treatment of cancer. However, the effects and underlying mechanism of 5-MF on IV-mediated ALI are still unclear. Here, we showed that 5-MF significantly improved the survival of mice with lethal IV infection and ameliorated IV-mediated lung edema, lung histological changes, and inflammatory cell lung recruitment. We found that 5-MF has antiviral activity against influenza A virus (IAV), which was probably associated with increased expression of radical S-adenosyl methionine domain containing 2 (RSAD2) and suppression of endosomal acidification. Moreover, IV-infected A549 cells with 5-MF treatment markedly reduced proinflammatory mediator expression (IL-6, CXCL8, TNF-α, CXCL10, CCL2, CCL3, CCL4, GM-CSF, COX-2, and PGE2) and prevented P-IKBα, P-P65, and P-P38 activation. Interestingly, we demonstrated that 5-MF treatment could trigger activation of AMP-activated protein kinase (AMPK)α in IV-infected A549 cells, as evidenced by activation of the AMPKα downstream molecule P53. Importantly, the addition of AMPKα blocker compound C dramatically abolished 5-MF-mediated increased levels of RSAD2, the inhibitory effects on H1N1 virus-elicited endosomal acidification, and the suppression expression of proinflammatory mediators (IL-6, TNF-α, CXCL10, COX-2 and PGE2), as well as the inactivation of P-IKBα, P-P65, and P-P38 MAPK signaling pathways. Furthermore, inhibition of AMPKα abrogated the protective effects of 5-MF on H1N1 virus-mediated lung injury and excessive inflammation in vivo. Taken together, these results indicate that 5-MF alleviated IV-mediated ALI and suppressed excessive inflammatory responses through activation of AMPKα signaling.

Seeds of Ginkgo biloba L. inhibit oxidative stress and inflammation induced by cigarette smoke in COPD rats through the Nrf2 pathway

Ethnopharmacological relevanceAs a traditional medicine, seeds of Ginkgo biloba L. (Gbs) have been used to treat cough or asthma for a long time. It is commonly used in clinic for lung diseases. However, its mechanism of lung protection is not completely clear. Aims of the studyThis research was designed to explore the protective effects of Gbs on antioxidant and inflammation during the chronic obstructive pulmonary disease (COPD) pathological process provoked by cigarette smoking (CS) in rats. Materials and methodsSix random groups including control group, CS model group, Gbs intervention groups (25 mg/kg, 50 mg/kg, and 100 mg/kg) and aminophylline group were composed of forty-eight rats. Smoking and intratracheal instillation of lipopolysaccharide (LPS) were used to establish the COPD rat model. Glutathione peroxidase (GSH-PX), malondialdehyde (MDA), superoxide dismutase (SOD), and enzyme-linked immunosorbent assay (ELISA) was used for quantifying the inflammatory factors such as IL-8, IL-6, IL-10, IL-17 and TNF-α. Western blotting were used for detecting the protein expressions of Nrf2, Keap1 and HO-1 in the lung tissues. ResultsGbs inhibits lung histological changes and decreased the inflammatory factors in both bronchoalveolar lavage fluid (BALF) and serum of CS-exposed rats, including IL-10, IL-17, IL-6, IL-8 and TNF-α. Gbs also inhibited the MDA level, increased SOD and GSH-PX activity in serum and changed expressions of Nrf2, Keap1 and HO-1 in the lung tissues. ConclusionGbs inhibit oxidative stress and inflammation induced by cigarette smoke in COPD rats through the Nrf2 Pathway.

Proteomic analysis and identification reveal the anti-inflammatory mechanism of clofazimine on lipopolysaccharide-induced acute lung injury in mice.

Background and objectiveAcute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) was increasingly recognized as one of the most severe acute hyperimmune response of coronavirus disease 2019 (COVID-19). Clofazimine (CFZ) has attracted attention due to its anti-inflammatory property in immune diseases as well as infectious diseases. However, the role and potential molecular mechanism of CFZ in anti-inflammatory responses remain unclear.MethodsWe analyze the protein expression profiles of CFZ and LPS from Raw264.7 macrophages using quantitative proteomics. Next, the protective effect of CFZ on LPS-induced inflammatory model is assessed, and its underlying mechanism is validated by molecular biology analysis.ResultsLC–MS/MS-based shotgun proteomics analysis identified 4746 (LPS) and 4766 (CFZ) proteins with quantitative information. The key proteins and their critical signal transduction pathways including TLR4/NF-κB/HIF-1α signaling was highlighted, which was involved in multiple inflammatory processes. A further analysis of molecular biology revealed that CFZ could significantly inhibit the proliferation of Raw264.7 macrophages, decrease the levels of TNF-α and IL-1β, alleviate lung histological changes and pulmonary edema, improve the survival rate, and down-regulate TLR4/NF-κB/HIF-1α signaling in LPS model.ConclusionThis study can provide significant insight into the proteomics-guided pharmacological mechanism study of CFZ and suggest potential therapeutic strategies for infectious disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-022-01623-w.

MiR-146a-5p accelerates sepsis through dendritic cell activation and glycolysis via targeting ATG7.

To unveil the role and regulatory mechanism of miR-146a-5p in sepsis. A sepsis cell model was established via lipopolysaccharide (LPS)-induction in dendritic cells (DCs). The maturation of DCs was evaluated via flow cytometry. Gene expression was measured through reverse-transcription quantitative polymerase chain reaction (RT-qPCR). The concentrations of inflammation biomarkers were revealed via enzyme-linked immunosorbent assay (ELISA). The pathological and histological changes in lungs in the sepsis mice model were analyzed via hematoxylin and eosin (H&E) staining. In this study,the miR-146a-5p level was elevated in the serum of sepsis patients and LPS-induced DCs but decreased in the serums of cured sepsis patients. Furthermore, miR-146a-5p deletion alleviated the activation of T cells and attenuated the imbalance of Th17/Treg. Besides, ATG7 was validated as a target of miR-146a-5p. ATG7 elevation enhanced lactate production and glucose uptake in LPS-triggered DCs. Additionally, upregulation of ATG7 suppressed the protein levels of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), phospho protein kinase B (p-AKT), and phosphorylated signal transducer and activator for transcription 3 (p-STAT3). In addition, miR-146a-5p downregulation alleviated T-cell activation, inflammation, lactate production, and glucose uptake in sepsis mice. Moreover, the lung injury due to sepsis was also attenuated as a result of miR-146a-5p silencing. MiR-146a-5p aggravates sepsis through DCs activation and glycolysis via targeting ATG7.

Innate Immune Responses to RSV Infection Facilitated by OGG1, an Enzyme Repairing Oxidatively Modified DNA Base Lesions

The primary cause of morbidity and mortality from infection with respiratory syncytial virus (RSV) is the excessive innate immune response(s) (IIR) in which reactive oxygen species (ROS) play key role(s). However, the mechanisms for these processes are not fully understood. We hypothesized that expressions of IIR genes are controlled by the ROS-generated epigenetic-like mark 7,8-dihydro-8-oxo(d)guanine (8-oxo(d)Gua) and 8-oxoguanine DNA glycosylase1 (OGG1). Here, we report that ROS not only generates intrahelical 8-oxo(d)Gua, but also enzymatically disables OGG1 in RSV-infected human airway epithelial cells and mouse lungs. OGG1 bound to 8-oxo(d)Gua in gene regulatory sequences promotes expression of IIR genes, and consequently exacerbates lung inflammation, histological changes, and body weight loss of experimental animals. Pharmacological inhibition of OGG1 substrate binding decreased expression of RSV-induced chemokine and cytokines and significantly lessened clinical symptoms. Results of mechanistic studies show that OGG1 binding at 8-oxo(d)Gua promoter regions modulated loading of transcription factors via transient cooperative interactions in RSV-infected lungs and airway epithelial cells. Other base specific DNA repair proteins had no effects. Collectively, this study identifies unprecedented roles of ROS-generated DNA base lesion(s) and cognate repair protein as a determinant of RSV-induced exuberant inflammation. Pharmaceutical inhibition of OGG1 interaction with its DNA substrate may represent a novel strategy in prevention/intervention of respiratory viral infections.

AKR1C1 alleviates LPS‑induced ALI in mice by activating the JAK2/STAT3 signaling pathway.

Acute lung injury (ALI) is a respiratory tract disease characterized by increased alveolar/capillary permeability, lung inflammation and structural damage to lung tissues, which can progress and transform into acute respiratory distress syndrome (ARDS). Although there are several treatment strategies available to manage this condition, there is still no specific cure for ALI. Aldo-keto reductase family 1 member C1 (AKR1C1) is a member of the aldo-keto reductase superfamily, and is a well-known Nrf2 target gene and an oxidative stress gene. The aim of the present study was to investigate the effects of AKR1C1 on a lipopolysaccharide (LPS)-induced ALI model. After mice received LPS treatment, the mRNA expression levels of AKR1C1 in the bronchoalveolar lavage fluid and serum were measured using reverse transcription-quantitative PCR and its relationship with the inflammatory factors and malondialdehyde levels were determined using correlation analysis. Next, AKR1C1 was overexpressed or knocked out in mice, and subsequently ALI was induced in mice using LPS. The severity of ALI, oxidative stress and inflammation in the lungs were measured, and the potential involvement of the Janus kinase 2 (JAK2)/signal transduction activator of transcription 3 (STAT3) signaling pathway was assessed by measuring the changes of lung injury parameters after the agonists of JAK2/STAT3 pathway, including interleukin (IL)-6 and colivelin, were administrated to mice. The results revealed that AKR1C1 expression was decreased in the LPS-induced ALI mouse model. AKR1C1 expression was inversely correlated with serum tumor necrosis factor-α, IL-6 and malondialdehyde levels, and positively correlated with serum IL-10 levels. AKR1C1 overexpression significantly attenuated lung injury, as shown by the changes in Evans blue leakage in the lung, lung wet/dry weight ratio, PaO2/FIO2 ratio, survival rate of mice and histological lung changes. In addition, the JAK2/STAT3 signaling pathway was significantly deactivated by AKR1C1+/+. When AKR1C1+/+ mice were treated with JAK2/STAT3 agonists, the effects of AKR1C1 overexpression on lung injury and oxidative stress were abolished. In conclusion, AKR1C1 may protect against oxidative stress and serve as a negative regulator of inflammation in ALI/ARDS. In addition, the JAK2/STAT3 signaling pathway could participate in the protective effects of AKR1C1 against ALI.

Cocculus hirsutus ameliorates gastric and lung injuries by suppressing Src/Syk

BackgroundCocculus hirsutus (L.) W. Thedo., a traditionally well-known plant, has confirmed antitumor properties as well as acute and chronic diuretic effects. However, little is known about its inflammatory activities and the potential effect on inflammatory disease treatment. PurposeOur aim in this study was to explore additional beneficial properties of C. hirsutus ethanol extract (Ch-EE) such as anti-inflammatory activity in vitro and in vivo as well as its underlying mechanisms and to provide a theoretical basis for its role as a candidate natural drug in clinical gastritis and lung disease therapy. Study DesignRAW264.7 cells, HEK293T cells, peritoneal macrophages, and mouse models of acute gastritis and acute lung injury were used to assess the anti-inflammatory activity of Ch-EE. MethodsDecreases in LPS-induced nitric oxide (NO) production and cytokine expression by RAW264.7 cells after Ch-EE treatment were evaluated by Griess assays and PCR, respectively. Transcription factor activity was assessed through luciferase reporter gene assay, and protein expression was determined by Western blotting analysis. Overexpression assays and cellular thermal shift assays were executed in HEK293T cells. Our two in vivo models were an HCl/EtOH-induced gastritis model and an LPS-induced lung injury model. Changes in stomach lesions, lung edema, and lung histology were examined upon treatment with Ch-EE. Components of Ch-EE were determined by liquid chromatography-mass spectrometry. ResultsLPS-induced nitric oxide production and Pam3CSK4- and L-NAME-induced NO production were inhibited by Ch-EE treatment of RAW264.7 cells. Furthermore, LPS-induced increases in transcript levels of iNOS, COX2, CCL12, and IL-1β were reduced by Ch-EE treatment. Ch-EE decreased both MyD88- and TRIF-induced NF-κB promotor activity. Proteins upstream of NF-κB, namely p-p50, p-p65, p-IκBα, p-AKT1, p-Src, and p-Syk, were all downregulated by Ch-EE. Moreover, Src and Syk were targets of Ch-EE. Ch-EE treatment reduced the size of inflammatory stomach lesions induced by HCl/EtOH, lung edema, and accumulation of activated neutrophils caused by LPS. ConclusionsThese results strongly suggest that Cocculus hirsutus can be developed as a promising anti-inflammatory remedy with Src- and Syk-inhibitory functions targeting diseases related to gastritis and lung injury.