Inhibits Lung Inflammation Research Articles

P-2028. Therapeutic Effect of Delayed Treatment with a Second-Generation 3CL Protease Inhibitor S-892216 in Hamsters Infected with SARS-CoV-2

Abstract Background SARS-CoV-2 and COVID-19 remain a major global health challenge. We have discovered a second-generation small molecule 3CL protease inhibitor S-892216 that is proceeding with phase 1 study in Japan. S-892216 has potent antiviral activity and addresses current treatment concerns of currently available antivirals regarding drug-drug interaction (DDI) and safety. We evaluated the therapeutic effect of S-892216 by using SARS-CoV-2 infected hamster model. In this study, virus titer in lungs and nasal turbinates (NT), lung inflammation, body weight loss and lung weight increase caused by SARS-CoV-2 infection were analyzed. Methods Syrian hamsters were intranasally inoculated with SARS-CoV-2 Omicron BE.1/BA.5-like. The hamsters were orally treated twice daily with S-892216 0.1, 1, and 10 mg/kg or ensitrelvir 50 mg/kg, or subcutaneously treated with nirmatrelvir 750/250 mg/kg (750 mg/kg for the loading dose and 250 mg/kg for the maintenance doses) from 1 day post infection for 5 days. Lungs and NT were collected for virus titers and lung inflammation evaluation. Lung weights were measured 7 days post infection and body weight changes were monitored until 14 days post infection. Results Oral treatment with S-892216 significantly reduced virus titers in lung (1.9- and 2.9-log reduction at the doses of 1 and 10 mg/kg, respectively) and NT (1.8- and 2.3-log reduction at the doses of 1 and 10 mg/kg, respectively) 3 days post infection. In addition, S-892216 suppressed lung inflammation and body weight loss caused by SARS-CoV-2 infection dose dependently. The virus titer reduction in lung and NT and suppression of lung inflammation and body weight losses were comparable in S-892216 1 mg/kg, ensitrelvir 50 mg/kg and nirmatrelvir 750/250 mg/kg treatment groups. Conclusion Oral treatment with S-892216 exhibited therapeutic effect in SARS-CoV-2 infected hamsters. Furthermore, S-892216 showed comparable efficacy at lower dosages compared to ensitrelvir and nirmatrelvir. These findings suggest that S-892216 could be used at lower dosage than the current treatment options in clinical settings. We will evaluate DDI and the safety potential of S-892216 in clinical trials in future. Disclosures Masaaki Nakashima, PhD, Shionogi&Co., Ltd.: Employee|Shionogi&Co., Ltd.: Stocks/Bonds (Private Company) Haruaki Nobori, PhD, Shionogi & Co., Ltd.: Employee|Shionogi & Co., Ltd.: Stocks/Bonds (Private Company) Alice Shimba, n/a, Shionogi & Co., Ltd.: Employee|Shionogi & Co., Ltd.: Stocks/Bonds (Private Company) Satoshi Miyagawa, n/a, Shionogi & Co., Ltd.: Employee|Shionogi & Co., Ltd.: Stocks/Bonds (Private Company) Akane Hayashi, n/a, Shionogi & Co., Ltd.: Stocks/Bonds (Private Company)|Shionogi TechnoAdvance Research: Employee Kazumi Matsumoto, n/a, Shionogi TechnoAdvance Research: Employee Kaoru Baba, n/a, Shionogi TechnoAdvance Research: Employee Keita Fukao, MD, SHIONOGI and CO., LTD.: Stocks/Bonds (Private Company)

Advances on physiology and pathology of subpopulations of macrophages in the lung tissue

Macrophages are vital in maintaining tissue homeostasis in the lungs by modulating and regulating immune responses. Based on different origins and anatomical locations, macrophages in the lungs are categorized into alveolar macrophages, interstitial macrophages, perivascular macrophages, and inflammatory macrophages. Alveolar macrophages are located in the alveolar spaces and are primarily responsible for maintaining alveolar surfactant homeostasis, defending against pathogens and regulating immune responses. Interstitial macrophages can maintain homeostasis, regulate immunity and anti-inflammation in the lung tissue. Perivascular macrophages play a crucial role in inhibiting lung inflammation, improving pulmonary fibrosis, and regulating lung tumor progression due to antigen-presenting and immunomodulatory effects. Inflammatory macrophages, which are differentiated from monocytes during inflammation, regulate the inflammatory process. This article reviews the origins of various subpopulations of macro-phages in the lung tissue and their physiological and pathological functions as well as discusses the underlying mechanisms and potential therapeutic targets.

Intratracheal Surfactant Administration Attenuates Hyperoxia-Induced Lung Injury and Fibrosis in Neonatal Rats

Abstract Background: Hyperoxia decreases surfactant production and suggests exogenous surfactant may be a potential treatment for hyperoxia-induced lung injury. This study aimed to investigate the effects of an animal-derived surfactant on hyperoxia-induced lung injury and fibrosis in newborn rats. Methods: Sprague Dawley rat pups were randomly reared either in room air (RA) or hyperoxic conditions (85% O2) from postnatal days 1–14. On postnatal day 4, the rats received an intratracheal injection of either 20 μL of normal saline (vehicle) or 20 μL of surfactant (Survanta). Our study included four study groups: RA + vehicle, RA + surfactant, 85% O2 + vehicle, and 85% O2 + surfactant. Body weights were recorded at birth and on postnatal days 4 and 14. On postnatal day 14, the lungs were dissected for histology, Western blotting, and cytokine measurements. Results: The hyperoxia-reared rats exhibited significantly higher lung injury scores, tumor necrosis factor-α (TNF-α) expression, transforming growth factor-β1 (TGF-β1) expression, and collagen deposition compared with the RA-reared rats. The surfactant alleviated hyperoxia-induced lung injury, inflammation, and fibrosis, as evidenced by the lower lung injury score, TNF-α expression, TGF-β1 expression, and collagen deposition in the lungs. Conclusion: The intratracheal administration of the surfactant ameliorated hyperoxia-induced lung injury and fibrosis and downregulated TNF-α and TGF-β1 expression, most likely by inhibiting lung inflammation and collagen deposition.

Exacerbation of influenza virus induced lung injury by alveolar macrophages and its suppression by pyroptosis blockade in a human lung alveolus chip.

Alveolar macrophages (AMs) are the major sentinel immune cells in human alveoli and play a central role in eliciting host inflammatory responses upon distal lung viral infection. Here, we incorporated peripheral human monocyte-derived macrophages within a microfluidic human Lung Alveolus Chip that recreates the human alveolar-capillary interface under an air-liquid interface along with vascular flow to study how residential AMs contribute to the human pulmonary response to viral infection. When Lung Alveolus Chips that were cultured with macrophages were infected with influenza H3N2, there was a major reduction in viral titers compared to chips without macrophages; however, there was significantly greater inflammation and tissue injury. Pro-inflammatory cytokine levels, recruitment of immune cells circulating through the vascular channel, and expression of genes involved in myelocyte activation were all increased, and this was accompanied by reduced epithelial and endothelial cell viability and compromise of the alveolar tissue barrier. These effects were partially mediated through activation of pyroptosis in macrophages and release of pro-inflammatory mediators, such as interleukin (IL)-1β, and blocking pyroptosis via caspase-1 inhibition suppressed lung inflammation and injury on-chip. These findings demonstrate how integrating tissue resident immune cells within human Lung Alveolus Chip can identify potential new therapeutic targets and uncover cell and molecular mechanisms that contribute to the development of viral pneumonia and acute respiratory distress syndrome (ARDS).

IRF3 inhibits inflammatory signaling pathways in macrophages to prevent viral pathogenesis.

Viral inflammation contributes to pathogenesis and mortality during respiratory virus infections. IRF3, a critical component of innate antiviral immune responses, interacts with pro-inflammatory transcription factor NF-κB, and inhibits its activity. This mechanism helps suppress inflammatory gene expression in virus-infected cells and mice. We evaluated the cells responsible for IRF3-mediated suppression of viral inflammation using newly engineered conditional Irf3Δ/Δ mice. Irf3Δ/Δ mice, upon respiratory virus infection, showed increased susceptibility and mortality. Irf3 deficiency caused enhanced inflammatory gene expression, lung inflammation, immunopathology, and damage, accompanied by increased infiltration of pro-inflammatory macrophages. Deletion of Irf3 in macrophages (Irf3MKO) displayed, similar to Irf3Δ/Δ mice, increased inflammatory responses, macrophage infiltration, lung damage, and lethality, indicating that IRF3 in these cells suppressed lung inflammation. RNA-seq analyses revealed enhanced NF-κB-dependent gene expression along with activation of inflammatory signaling pathways in infected Irf3MKO lungs. Targeted analyses revealed activated MAPK signaling in Irf3MKO lungs. Therefore, IRF3 inhibited inflammatory signaling pathways in macrophages to prevent viral inflammation and pathogenesis.

Feruloylated Oligosaccharides Prevented Influenza-Induced Lung Inflammation via the RIG-I/MAVS/TRAF3 Pathway.

Uncontrolled inflammation contributes significantly to the mortality in acute respiratory infections. Our previous research has demonstrated that maize bran feruloylated oligosaccharides (FOs) possess notable anti-inflammatory properties linked to the NF-kB pathway regulation. In this study, we clarified that the oral administration of FOs moderately inhibited H1N1 virus infection and reduced lung inflammation in influenza-infected mice by decreasing a wide spectrum of cytokines (IFN-α, IFN-β, IL-6, IL-10, and IL-23) in the lungs. The mechanism involves FOs suppressing the transduction of the RIG-I/MAVS/TRAF3 signaling pathway, subsequently lowering the expression of NF-κB. In silico analysis suggests that FOs have a greater binding affinity for the RIG-I/MAVS signaling complex. This indicates that FOs have potential as promising targets for immune modulation. Moreover, in MAVS knockout mice, we confirmed that the anti-inflammatory function of FOs against influenza depends on MAVS. Comprehensive analysis using 16S rRNA gene sequencing and metabolite profiling techniques showed that FOs have the potential to restore immunity by modulating the gut microbiota. In conclusion, our study demonstrates that FOs are effective anti-inflammatory phytochemicals in inhibiting lung inflammation caused by influenza. This suggests that FOs could serve as a potential nutritional strategy for preventing the H1N1 virus infection and associated lung inflammation.

Bufei Huoxue capsule attenuates COPD-related inflammation and regulates intestinal microflora, metabolites.

Background: Bufei Huoxue capsule (BFHX) is widely used for the clinical treatment of chronic obstructive pulmonary disease (COPD) in China. Objectives: The aim of this study is to explore the effects on COPD and the underlying mechanism of BFHX. The process and methods: In this study, we established a COPD mouse model through cigarette smoke (CS) exposure in combination with lipopolysaccharide (LPS) intratracheal instillation. Subsequently, BFHX was orally administrated to COPD mice, and their pulmonary function, lung pathology, and lung inflammation, including bronchoalveolar lavage fluid (BALF) cell count and classification and cytokines, were analyzed. In addition, the anti-oxidative stress ability of BFHX was detected by Western blotting, and the bacterial diversity, abundance, and fecal microbiome were examined using 16S rRNA sequencing technology. Outcome: BFHX was shown to improve pulmonary function, suppress lung inflammation, decrease emphysema, and increase anti-oxidative stress, whereas 16S rRNA sequencing indicated that BFHX can dynamically regulate the diversity, composition, and distribution of the intestinal flora microbiome and regulate the lysine degradation and phenylalanine metabolism of COPD mice. These results highlight another treatment option for COPD and provide insights into the mechanism of BFHX.

Short-Chain Fatty Acid (SCFA) as a Connecting Link between Microbiota and Gut-Lung Axis-A Potential Therapeutic Intervention to Improve Lung Health.

The microbiome is an integral part of the human gut, and it plays a crucial role in the development of the immune system and homeostasis. Apart from the gut microbiome, the airway microbial community also forms a distinct and crucial part of the human microbiota. Furthermore, several studies indicate the existence of communication between the gut microbiome and their metabolites with the lung airways, called "gut-lung axis". Perturbations in gut microbiota composition, termed dysbiosis, can have acute and chronic effects on the pathophysiology of lung diseases. Microbes and their metabolites in lung stimulate various innate immune pathways, which modulate the expression of the inflammatory genes in pulmonary leukocytes. For instance, gut microbiota-derived metabolites such as short-chain fatty acids can suppress lung inflammation through the activation of G protein-coupled receptors (free fatty acid receptors) and can also inhibit histone deacetylase, which in turn influences the severity of acute and chronic respiratory diseases. Thus, modulation of the gut microbiome composition through probiotic/prebiotic usage and fecal microbiota transplantation can lead to alterations in lung homeostasis and immunity. The resulting manipulation of immune cells function through microbiota and their key metabolites paves the way for the development of novel therapeutic strategies in improving the lung health of individuals affected with various lung diseases including SARS-CoV-2. This review will shed light upon the mechanistic aspect of immune system programming through gut and lung microbiota and exploration of the relationship between gut-lung microbiome and also highlight the therapeutic potential of gut microbiota-derived metabolites in the management of respiratory diseases.

Danshensu inhibits SARS-CoV-2 by targeting its main protease as a specific covalent inhibitor and discovery of bifunctional compounds eliciting antiviral and anti-inflammatory activity

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to human. Since there are still no effective treatment options against the new emerging variants of SARS-CoV-2, it is necessary to devote a continuous endeavor for more targeted drugs and the preparation for the next pandemic. Salvia miltiorrhiza and its active ingredients possess wide antiviral activities, including against SARS-CoV-2. Danshensu, as one of the most important active ingredients in Salvia miltiorrhiza, has been reported to inhibit the entry of SARS-CoV-2 into ACE2 (angiotensin-converting enzyme 2)-overexpressed HEK-293T cells and Vero-E6 cells. However, there is a paucity of information regarding its detailed target and mechanism against SARS-CoV-2. Here, we present Danshensu as a covalent inhibitor of 3-chymotrypsin-like protease (3CLpro) against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. Further molecular docking, site-directed mutagenesis, circular dichroism (CD) and fluorescence spectra revealed that Danshensu covalently binds to C145 of SARS-CoV-2 3CLpro, meanwhile forming the hydrogen bonds with S144, H163 and E166 in the S1 site. Structure-based optimization of Danshensu led to the discovery of the promising compounds with good inhibitory activity and microsomal stability in vitro. Due to Danshensu inhibiting lung inflammation in the mouse model, we found that Danshensu derivatives also showed better anti-inflammatory activity than Danshensu in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Thus, our study provides not only the clue of the efficacy of Salvia miltiorrhiza against SARS-CoV-2, but also a detailed mechanistic insight into the covalent mode of action of Danshensu for design of covalent inhibitors against SARS-CoV-2 3CLpro, highlighting its potential as a bifunctional molecule with antivirus and anti-inflammation.

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

The water-soluble subfraction from Artemisia argyi alleviates LPS-induced inflammatory responses via multiple pathways and targets in vitro and in vivo

Ethnopharmacological relevanceAs a traditional Chinese medicine, Artemisia argyi has been used medicinally and eaten for more than 2000 years in China. It is widely reported in treating inflammatory diseases such as eczema, dermatitis, arthritis, allergic asthma and colitis. Although several studies claim that its volatile oil and organic reagent extracts have certain anti-inflammatory effects, the water-soluble fractions and molecular mechanisms have not been studied. Aim of the studyTo evaluate the therapeutic effect of A. argyi water extract (AAWE) on lipopolysaccharide (LPS)-induced inflammatory responses and to identify the most effective water-soluble subfractions. Moreover, the relevant pharmacological and molecular mechanisms by which the active subfraction mitigates inflammation were further investigated. Materials and methodsFirstly, RAW 264.7 cells stimulated with LPS were treated with AAWE (50, 100, and 200 μg/mL) or the water-soluble subfractions separated by D101 macroporous resin (AAWE1-AAWE4, 100 μg/mL), and NO production and mRNA levels of inflammatory genes were evaluated to determine the most effective water-soluble subfractions. Secondly, the chemical components of the active subfraction (AAWE4) were analyzed by UPLC-QTOF-MS. Thirdly, transcriptome and network pharmacology analysis, RT-qPCR and Western blotting assays were conducted to explore the underlying anti-inflammatory mechanism and active compounds of AAWE4. Subsequently, the binding ability of the potential active components in AAWE4 to the core targets was further determined by molecular docking. Eventually, the in vivo anti-inflammatory activity of AAWE4 (1.17, 2.34 and 4.68 g/kg, administered per day for 7 d) was evaluated in mice with LPS-induced systemic inflammation. ResultsIn this study, AAWE showed excellent anti-inflammatory effects, and its water-soluble subfraction AAWE4 exhibited the strongest inhibitory effect on NO concentration and inflammatory gene mRNA expression after LPS stimulation, indicating that it was the most effective subfraction. Thereafter, four main compounds in AAWE4 were confirmed or tentatively identified by UPLC-QTOF-MS, including three flavonoid glycosides and one phenolic acid. Furthermore, the transcriptome and network pharmacology analysis showed that AAWE4 inhibited inflammation via multiple pathways and multiple targets. Based on the RT-qPCR and Western blotting results, AAWE4 downregulated not only the p38, PI3K, CCL5, MMP9, AP-1, and BCL3 mRNA expression levels activated by LPS but also their upstream and downstream protein expression levels and protein phosphorylation (p-AKT/AKT, p-p38/p38, p-ERK/ERK, p-JNK/JNK). Moreover, four identified compounds (isochlorogenic acid A, vicenin-2, schaftoside and isoschaftoside) could significantly inhibit NO content and the overexpression of inflammatory factors TNF-α, IL-1β, iNOS and COX-2 mRNA induced by LPS, and the molecular docking confirmed the high binding activity of four active compounds with selected core targets (p38, AKT1, MMP9, and CCL5). In addition, the mRNA expression and immunohistochemical analysis showed that AAWE44 could inhibit lung inflammation via multiple pathways and multiple targets in vivo. ConclusionsThe findings of this study suggest that the water-soluble subfraction AAWE4 from A. argyi ameliorated the inflammation caused by LPS through multiple pathways and multiple targets in vitro and in vivo, providing scientific support for the medicinal use of A. argyi. Importantly, it shows that the A. argyi subfraction AAWE4 can be developed as an anti-inflammatory drug.

Alismol Purified from the Tuber of Alisma orientale Relieves Acute Lung Injury in Mice via Nrf2 Activation.

Since the ethanol extract of Alisma orientale Juzepzuk (EEAO) suppresses lung inflammation by suppressing Nuclear Factor-kappa B (NF-κB) and activating Nuclear Factor Erythroid 2-related Factor 2 (Nrf2), we set out to identify chemicals constituting EEAO that suppress lung inflammation. Here, we provide evidence that among the five most abundant chemical constituents identified by Ultra Performance Liquid Chromatography (UPLC) and Nuclear Magnetic Resonance (NMR), alismol is one of the candidate constituents that suppresses lung inflammation in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model and protects mice from ALI-like symptoms. Alismol did not induce cytotoxicity or reactive oxygen species (ROS). When administered to the lung of LPS-induced ALI mice (n = 5/group), alismol decreased the level of neutrophils and of the pro-inflammatory molecules, including Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Monocyte Chemoattractant Protein-1 (MCP-1), Interferon-gamma (IFN-γ), and Cyclooxygenase-2 (COX-2), suggesting an anti-inflammatory activity of alismol. Consistent with these findings, alismol ameliorated the key features of the inflamed lung of ALI, such as high cellularity due to infiltrated inflammatory cells, the development of hyaline membrane structure, and capillary destruction. Unlike EEAO, alismol did not suppress NF-κB activity but rather activated Nrf2. Consequently, alismol induced the expression of prototypic genes regulated by Nrf2, including Heme Oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase-1 (NQO-1), and glutamyl cysteine ligase catalytic units (GCLC). Alismol activating Nrf2 appears to be associated with a decrease in the ubiquitination of Nrf2, a key suppressive mechanism for Nrf2 activity. Together, our results suggest that alismol is a chemical constituent of EEAO that contributes at least in part to suppressing some of the key features of ALI by activating Nrf2.

Knockdown of PHLDA1 alleviates sepsis-induced acute lung injury by downregulating NLRP3 inflammasome activation.

To investigate the regulatory mechanism of pleckstrin homology-like domain, family A, member 1 (PHLDA1) in sepsis-induced acute lung injury (ALI). Mice model of sepsis were established by cecal ligation and puncture (CLP). The expression of PHLDA1 was reduced by injecting short hairpin RNA (shRNA)-PHLDA1 into the tail vein. The levels of PHLDA1, pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1β, IL-18, super-oxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), molecular mechanism related to pyroptosis, such as caspase 1, adaptor apoptosis-associated speck-like protein containing a CARD (ASC), and gasdermin D (GSDMD)-N, and nucleotide oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) were tested by Western blot analysis, quantitative real-time polymerase chain reaction, and enzyme-linked-immunosorbent serologic assay. Pathological changes in lung tissues were examined by hematoxylin and eosin staining. Wet-dry weight ratio of lung tissues was observed. The expression of PHLDA1 was up-regulated in lung tissues from CLP-induced septic mice. Knockdown of PHLDA1 could reduce lung injury and wet-dry weight ratio in mice with sepsis-induced ALI. Moreover, silencing of PHLDA1 decreased the expressions of IL-1β, TNF-α, IL-18, IL-6, and MDA but increased SOD and GSH expressions in CLP-induced septic mice. The expressions of NLRP3, GSDMD-N, ASC, and caspase 1 were decreased by PHLDA1 silencing. Knockdown of PHLDA1 inhibited lung inflammation and pyroptosis in mice with sepsis-induced ALI by down-regulating NLRP3.

Efficiency of exogenous surfactant combined with intravenous N-acetylcysteine in two-hit rodent model of ARDS

Accumulation of reactive oxygen species during hyperoxia together with secondary bacteria-induced inflammation leads to lung damage in ventilated critically ill patients. Antioxidant N-acetylcysteine (NAC) in combination with surfactant may improve lung function. We compared the efficacy of NAC combined with surfactant in the double-hit model of lung injury. Bacterial lipopolysaccharide (LPS) instilled intratracheally and hyperoxia were used to induce lung injury in Wistar rats. Animals were mechanically ventilated and treated intravenously with NAC alone or in combination with intratracheal surfactant (poractant alfa; PSUR+NAC). Control received saline. Lung functions, inflammatory markers, oxidative damage, total white blood cell (WBC) count and lung oedema were evaluated during 4 hrs. Administration of NAC increased total antioxidant capacity (TAC) and decreased IL-6. This effect was potentiated by the combined administration of surfactant and NAC. In addition, PSUR+NAC reduced the levels of TNFα, IL-1ß, and TAC compared to NAC only and improved lung injury score. The combination of exogenous surfactant with NAC suppresses lung inflammation and oxidative stress in the experimental double-hit model of lung injury.

To Analyze the Mechanism of Glycyrrhiza uralensis (Licorice) in the Treatment of COVID-19 Based on Network Pharmacology and Molecular Docking Technology

The current study, the effectiveness of analyzing Glycyrrhiza uralensis for the treatment of COVID-19 was investigated using an integrated network pharmacology and molecular docking approach. Through network pharmacology, establishment of Protein-Protein Interactions (PPI), molecular docking simulations, GO analysis, and KEGG analysis, the aim was to investigate the mechanism of licorice in the treatment of COVID-19. There are 7 core genes corresponding to 3 bioactive compounds of G. uralensis. The target proteins of G. uralensis for the treatment of neocoronary pneumonia could be enriched to cancer pathway, lipid The target proteins of G. uralensis for the treatment of neocoronary pneumonia could be enriched to cancer pathway, lipid and atherosclerosis pathway, PI3K-Akt signaling pathway, and down-regulate the activity of core targets to inhibit the expression of virus SARS-CoV-2. Based on molecular docking simulations, the largest significant binding affinities were found for TNF and 7-Methoxy-2-methyl isoflavone (-6.95 kcal/mol), MAPK1 and kaempferol (-6.75 kcal/mol), and AKT1 and quercetin (-6.74 kcal/mol). Quercetin induces viral cell cycle arrest and inhibits growth and metastasis by engaging in the induction and expression of key intracellular targets. The flavonoid chemicals of kaempferol can inhibit inflammation-related signaling pathways and suppress the release of inflammation-related factors, and 7-methoxy-2-methylisoflavone may have therapeutic effects on COVID-19 by reducing hydroxyproline levels to suppress lung inflammation and fibrosis indices and modulate immune function. This suggests that G. uralensis has an interfering effect on novel coronaviruses and its main active component has a strong binding ability to the core gene of SARS-CoV-2, providing knowledge for future studies based on COVID-19.

Alpha-ketoglutarate supplementation reduces inflammation and thrombosis in type 2 diabetes by suppressing leukocyte and platelet activation.

The interplay between platelets and leukocytes contributes to the pathogenesis of inflammation, thrombosis, and cardiovascular diseases (CVDs) in type 2 diabetes (T2D). Our recent studies described alpha-ketoglutarate (αKG), a Krebs cycle intermediate metabolite as an inhibitor to platelets and leukocytes activation by suppressing phosphorylated-Akt (pAkt) through augmentation of prolyl hydroxylase-2 (PHD2). Dietary supplementation with a pharmacological concentration of αKG significantly inhibited lung inflammation in mice with either SARS-CoV-2 infection or exposed to hypoxia treatment. We therefore investigated if αKG supplementation could suppress hyperactivation of these blood cells and reduce thromboinflammatory complications in T2D. Our study describes that dietary supplementation with αKG (8 mg/100 g body wt. daily) for 7 days significantly reduced the activation of platelets and leukocytes (neutrophils and monocytes), and accumulation of IL1β, TNFα, and IL6 in peripheral blood of T2D mice. αKG also reduced the infiltration of platelets and leukocytes, and accumulation of inflammatory cytokines in lungs by suppressing pAkt and pP65 signaling. In a cross-sectional investigation, our study also described the elevated platelet-leukocyte aggregates and pro-inflammatory cytokines in circulation of T2D patients. T2D platelets and leukocytes showed an increased aggregation and thrombus formation in vitro. Interestingly, a pre-incubation of T2D blood samples with octyl αKG significantly suppressed the activation of these blood cells and ameliorated aggregate/thrombus formation in vitro. Thus, suggesting a potential therapeutic role of αKG against inflammation, thrombosis, and CVDs in T2D.

Autophagy benefits the in vitro and in vivo clearance of Talaromyces marneffei

Talaromycosis, namely Talaromyces marneffei infection, is increasing gradually and has a high mortality rate even under antifungal therapy. Although autophagy acts differently on different pathogens, it is a promising therapeutic strategy. However, information on autophagy in macrophages and animals upon infection by T. marneffei is still limited. Therefore, several models were employed here to investigate the role of autophagy in host defense against T. marneffei, including RAW264.7 macrophages as in vitro models, different types of Caenorhabditis elegans and BALB/c mice as in vivo models. We applied the clinical T. marneffei isolate SUMS0152 in this study. T. marneffei-infected macrophages exhibit increased formation of autophagosomes. Further, macrophage autophagy promoted by rapamycin or Earle's balanced salt solution (EBSS) inhibited the viability of intracellular T. marneffei. In vivo, compared with uninfected Caenorhabditis elegans, the wild-type nematodes upregulated the expression of the autophagy-related gene lgg-1 and atg-18, and nematodes carrying GFP reporter were induced to form autophagosomes (GFP::LGG-1) after T. marneffei infection. Furthermore, the knockdown of lgg-1 significantly reduced the survival rate of T. marneffei-infected nematodes. Likewise, the autophagy activator rapamycin reduced the fungal burden and suppressed lung inflammation in a mouse model of infection. In conclusion, autophagy is essential for host defense against T. marneffei in vitro and in vivo. Therefore, autophagy may be an attractive target for developing new therapeutics to treat talaromycosis.

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Anti-PPIL-5 antibody exacerbates disease of severe influenza in a murine experimental model

Abstract Respiratory viruses such as SARS-CoV2 cause serious emerging infectious diseases. Even seasonal influenza results in significant morbidity and mortality. Host immunity responds to influenza virus infection to eradicate the pathogen. However, dysregulated responses such as the cytokine storm contribute to severe disease. Mechanistic dissection of immune regulation may offer practical solutions for severe influenza. In our mouse model of severe influenza, a LAG-3 regulatory response evolves with the pro-inflammatory IFN-γ response in influenza virus hemagglutinin (HA)-specific CD4+ T cells. Adoptive transfer of LAG-3(+) IFN-γ(-) HA-specific CD4+ T cells suppressed lung inflammation without compromised virus eradication. Genomic study of LAG-3(+) IFN-γ(−) CD4+ T cells revealed up-regulation of the PPIL-5 [Peptidylprolyl isomerase (Cyclophilin)-like 5] gene. PPIL-5(+) HA-specific CD4+ T cells accumulated in the lung with time after infection. PPIL-5 protein suppressed HA-specific CD4+ T cell activation in vitro in a dose dependent manner. PPIL-5 blockade in vitro with siRNA reversed immune suppression and unleashed activation of HA-specific CD4+ T cells. PPIL-5 blockade in vivo with monoclonal anti-PPIL-5 antibodies attenuated LAG-3 and PD-1 and augmented IFN-γ responses of the lung-infiltrating HA-specific CD4+ T cells, with aggravated disease and intensified mortality of severe influenza. The HA-specific CD4+ T cells took less glucose and produced lactate with anti-PPIL-5 blockade. They adopted anaerobic glycolysis in this reverted inflammatory response. Our results suggest that PPIL-5, similar to LAG-3, is also an immune checkpoint and a possible target to be manipulated for alleviation of severe influenza. The Research Center for Emerging Viral Infections receives support from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan and NSTC 111-2634-F-182-001 from the National Science and Technology Council, Taiwan. This work was supported by Projects MOST 111-2314-B-182-029 (AD) and MOST-105-2321-B-182A-003-MY3 (AD) from the National Science and Technology Council, Taiwan, and by CMRPG3L1671 (CTH), CMRPVVJ0052 (CTH), and CMRPG3J1771 (CYH) from the Medical Research Project Fund, Chang Gung Memorial Hospital, Taiwan.

Modified Dingchuan Decoction treats cough-variant asthma by suppressing lung inflammation and regulating the lung microbiota.

Modified Dingchuan Decoction (MDD) is a Chinese medicine formula containing 11 materials with cough suppression, asthma relief, and anti-inflammatory effects. This study aimed to evaluate the therapeutic effect of MDD on cough-variant asthma (CVA) and to investigate its mechanism of action. The chemical constituents of MDD were analyzed by ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). A guinea pig CVA model was established using an intramuscular injection of ovalbumin (OVA), combined with an intraperitoneal injection of aluminum hydroxide [Al(OH)3] and nebulized OVA. At the beginning of day 18, the low, medium, and high MDD groups were gavaged with 7.23g/kg, 14.46g/kg, and 28.92g/kg of MDD, respectively, and the positive group was gavaged with 5mg/kg of prednisone acetate combined with 1mg/kg of montelukast sodium; the normal and model groups were given an equal volume of distilled water, once a day for 21 days. The cough was induced by 10-3mol/L capsaicin solution 1h after the last administration, and the number of coughs and the latency of coughs were evaluated. Hematoxylin and eosin staining (H&E) was used to observe pathological changes in the lungs and airways. The concentration of inflammatory factors in bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). We analyzed the lung microbiota using 16S ribosomal DNA (16S rDNA) high-throughput sequencing. The 38 chemical components were found in MDD, and MDD reduced the number of coughs in guinea pigs with CVA, prolonged cough latency, improved pathological damage to the lungs and airways, regulated inflammatory factor levels in BALF, and modulated the lung microbiota. This study demonstrated that treating CVA with MDD may be related to inhibiting lung inflammation and regulating lung microbiota.