Orally administered BZL-sRNA-20 oligonucleotide targeting TLR4 effectively ameliorates acute lung injury in mice.

The global COVID-19 pandemic emerged at the end of December 2019. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common lethal outcomes of bacterial lipopolysaccharide (LPS), avian influenza virus, and SARS-CoV-2. Toll-like receptor 4 (TLR4) is a key target in the pathological pathway of ARDS and ALI. Previous studies have reported that herbal small RNAs (sRNAs) are a functional medical component. BZL-sRNA-20 (Accession number: B59471456; Family ID: F2201.Q001979.B11) is a potent inhibitor of Toll-like receptor 4 (TLR4) and pro-inflammatory cytokines. Furthermore, BZL-sRNA-20 reduces intracellular levels of cytokines induced by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (poly (I:C)). We found that BZL-sRNA-20 rescued the viability of cells infected with avian influenza H5N1, SARS-CoV-2, and several of its variants of concern (VOCs). Acute lung injury induced by LPS and SARS-CoV-2 in mice was significantly ameliorated by the oral medical decoctosome mimic (bencaosome; sphinganine (d22:0)+BZL-sRNA-20). Our findings suggest that BZL-sRNA-20 could be a pan-anti-ARDS ALI drug.

Core genes involved in the regulation of acute lung injury and their association with COVID-19 and tumor progression: A bioinformatics and experimental study.

Acute lung injury (ALI) is a specific form of lung damage caused by different infectious and non-infectious agents, including SARS-CoV-2, leading to severe respiratory and systemic inflammation. To gain deeper insight into the molecular mechanisms behind ALI and to identify core elements of the regulatory network associated with this pathology, key genes involved in the regulation of the acute lung inflammatory response (Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Ptx3, Socs3) were revealed using comprehensive bioinformatics analysis of whole-genome microarray datasets, functional annotation of differentially expressed genes (DEGs), reconstruction of protein-protein interaction networks and text mining. The bioinformatics data were validated using a murine model of LPS-induced ALI; changes in the gene expression patterns were assessed during ALI progression and prevention by anti-inflammatory therapy with dexamethasone and the semisynthetic triterpenoid soloxolone methyl (SM), two agents with different mechanisms of action. Analysis showed that 7 of 8 revealed ALI-related genes were susceptible to LPS challenge (up-regulation: Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Socs3; down-regulation: Cat) and their expression was reversed by the pre-treatment of mice with both anti-inflammatory agents. Furthermore, ALI-associated nodal genes were analysed with respect to SARS-CoV-2 infection and lung cancers. The overlap with DEGs identified in postmortem lung tissues from COVID-19 patients revealed genes (Saa1, Rsad2, Ifi44, Rtp4, Mmp8) that (a) showed a high degree centrality in the COVID-19-related regulatory network, (b) were up-regulated in murine lungs after LPS administration, and (c) were susceptible to anti-inflammatory therapy. Analysis of ALI-associated key genes using The Cancer Genome Atlas showed their correlation with poor survival in patients with lung neoplasias (Ptx3, Timp1, Serpine1, Plaur). Taken together, a number of key genes playing a core function in the regulation of lung inflammation were found, which can serve both as promising therapeutic targets and molecular markers to control lung ailments, including COVID-19-associated ALI.

Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury.

qRT-PCR still remains the most widely used method for quantifying gene expression levels, although newer technologies such as next generation sequencing are becoming increasingly popular. A critical, yet often underappreciated, problem when analysing qRT-PCR data is the selection of suitable reference genes. This problem is compounded in situations where up to 25% of all genes may change (e.g., due to leukocyte invasion), as is typically the case in ARDS. Here, we examined 11 widely used reference genes for their suitability in commonly used models of acute lung injury (ALI): ventilator-induced lung injury (VILI), in vivo and ex vivo, lipopolysaccharide plus mechanical ventilation (MV), and hydrochloric acid plus MV. The stability of reference gene expression was determined using the NormFinder, BestKeeper, and geNorm algorithms. We then proceeded with the geNorm results because this is the only algorithm that provides the number of reference genes required to achieve normalisation. We chose interleukin-6 (Il-6) and C-X-C motif ligand 1 (Cxcl-1) as the genes of interest to analyse and demonstrate the impact of inappropriate normalisation. Reference gene stability differed between the ALI models and even within the subgroup of VILI models, no common reference gene index (RGI) could be determined. NormFinder, BestKeeper, and geNorm produced slightly different, but comparable results. Inappropriate normalisation of Il-6 and Cxcl1 gene expression resulted in significant misinterpretation in all four ALI settings. In conclusion, choosing an inappropriate normalisation strategy can introduce different kinds of bias such as gain or loss as well as under- or overestimation of effects, affecting the interpretation of gene expression data.

Molecular Mechanisms of Vascular Damage During Lung Injury.

A variety of pulmonary and systemic insults promote an inflammatory response causing increased vascular permeability, leading to the development of acute lung injury (ALI), a condition necessitating hospitalization and intensive care, or the more severe acute respiratory distress syndrome (ARDS), a disease with a high mortality rate. Further, COVID-19 pandemic-associated ARDS is now a major cause of mortality worldwide. The pathogenesis of ALI is explained by injury to both the vascular endothelium and the alveolar epithelium. The disruption of the lung endothelial and epithelial barriers occurs in response to both systemic and local production of pro-inflammatory cytokines. Studies that evaluate the association of genetic polymorphisms with disease risk did not yield many potential therapeutic targets to treat and revert lung injury. This failure is probably due in part to the phenotypic complexity of ALI/ARDS, and genetic predisposition may be obscured by the multiple environmental and behavioral risk factors. In the last decade, new research has uncovered novel epigenetic mechanisms that control ALI/ARDS pathogenesis, including histone modifications and DNA methylation. Enzyme inhibitors such as DNMTi and HDACi may offer new alternative strategies to prevent or reverse the vascular damage that occurs during lung injury. This review will focus on the latest findings on the molecular mechanisms of vascular damage in ALI/ARDS, the genetic factors that might contribute to the susceptibility for developing this disease, and the epigenetic changes observed in humans, as well as in experimental models of ALI/ADRS.

Hypoxia induces expression of angiotensin-converting enzyme II in alveolar epithelial cells: Implications for the pathogenesis of acute lung injury in COVID-19.

SARS-CoV-2 uptake by lung epithelial cells is a critical step in the pathogenesis of COVID-19. Viral entry is dependent on the binding of the viral spike protein to the angiotensin converting enzyme II protein (ACE2) on the host cell surface, followed by proteolytic cleavage by a host serine protease such as TMPRSS2. Infection of alveolar epithelial cells (AEC) in the distal lung is a key feature in progression to the acute respiratory distress syndrome (ARDS). We hypothesized that AEC expression of ACE2 is induced by hypoxia. In a murine model of hypoxic stress (12% FiO2), the total lung Ace2 mRNA and protein expression was significantly increased after 24 hours in hypoxia compared to normoxia (21% FiO2). In experiments with primary murine type II AEC, we found that exposure to hypoxia either in vivo (prior to isolation) or in vitro resulted in greatly increased AEC expression of both Ace2 (mRNA and protein) and of Tmprss2. However, when isolated type II AEC were maintained in culture over 5 days, with loss of type II cell characteristics and induction of type I cell features, Ace2 expression was greatly reduced, suggesting that this expression was a feature of only this subset of AEC. Finally, in primary human small airway epithelial cells (SAEC), ACE2 mRNA and protein expression were also induced by hypoxia, as was binding to purified spike protein. Hypoxia-induced increase in ACE2 expression in type II AEC may provide an explanation of the extended temporal course of human patients who develop ARDS in COVID-19.

Ethnic and age-specific acute lung injury/acute respiratory distress syndrome risk associated with angiotensin-converting enzyme insertion/deletion polymorphisms, implications for COVID-19: A meta-analysis.

BACKGROUND: The reported association between an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene and the risk for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) remains controversial despite the publication of four meta-analyses on this topic. Here, we updated the meta-analysis with more studies and additional assessments that include adults and children within the context of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Sixteen articles (22 studies) were included. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using three genetic models (allele, recessive and dominant), in which ARDS patients were compared with non-ARDS patients (A1) and healthy controls (A2). Mortality outcomes were also assessed (A3). The influence of covariates was examined by meta-regression. Bonferroni correction was performed for multiple pooled associations. Subgroup analyses based on ethnicity (Asians, Caucasians) and life stage (adults, children) were conducted. Heterogeneity was addressed with outlier treatment. RESULTS: This meta-analysis generated 68 comparisons, 21 of which were significant. Of the 21, four A1 and three A3 highly significant (Pa = 0.00001-0.0008) outcomes withstood Bonferroni correction. For A1, allele and recessive associations were found in overall (OR 0.49, 95% CI 0.39-0.61), Caucasians (OR 0.46, 95% CI 0.35-0.61) and children (ORs 0.49-0.66, 95% CI 0.33-0.84) analyses. For A3, associations were found in overall (dominant: OR 0.45, 95% CI 0.29-0.68) and Asian subgroup (allele/ dominant: ORs 0.31-0.39, 95% CIs 0.18-0.63) analyses. These outcomes were either robust, or statistically powered or both and uninfluenced by covariates. CONCLUSIONS: Significant associations of the ACE I/D polymorphism with the risk of ALI/ARDS were indicated in Caucasians and children as well as in Asians in mortality analysis. These findings were underpinned by high significance, high statistical power and robustness. ACE genotypes may be useful for ALI/ARDS therapy for patients with COVID-19.

Oestrogen-mediated upregulation of the Mas receptor contributes to sex differences in acute lung injury and lung vascular barrier regulation.

Epidemiological data from the SARS-CoV-2 outbreak suggest sex differences in mortality and vulnerability; however, sex-dependent incidence of acute respiratory distress syndrome (ARDS) remains controversial and the sex-dependent mechanisms of endothelial barrier regulation are unknown. In premenopausal women, increased signalling of angiotensin (Ang)(1-7) via the Mas receptor has been linked to lower cardiovascular risk. Since stimulation of the Ang(1-7)/Mas axis protects the endothelial barrier in acute lung injury (ALI), we hypothesised that increased Ang(1-7)/Mas signalling may protect females over males in ALI/ARDS.Clinical data were collected from Charité inpatients (Berlin) and sex differences in ALI were assessed in wild-type (WT) and Mas-receptor deficient (Mas-/- ) mice. Endothelial permeability was assessed as weight change in isolated lungs and as transendothelial electrical resistance (TEER) in vitroIn 734 090 Charité inpatients (2005-2016), ARDS had a higher incidence in men as compared to women. In murine ALI, male WT mice had more lung oedema, protein leaks and histological evidence of injury than female WT mice. Lung weight change in response to platelet-activating factor (PAF) was more pronounced in male WT and female Mas-/- mice than in female WT mice, whereas Mas-receptor expression was higher in female WT lungs. Ovariectomy attenuated protection in female WT mice and reduced Mas-receptor expression. Oestrogen increased Mas-receptor expression and attenuated endothelial leakage in response to thrombin in vitro This effect was alleviated by Mas-receptor blockade.Improved lung endothelial barrier function protects female mice from ALI-induced lung oedema. This effect is partially mediated via enhanced Ang(1-7)/Mas signalling as a result of oestrogen-dependent Mas expression.

An integrated network pharmacology and RNA-Seq approach for exploring the preventive effect of Lonicerae japonicae flos on LPS-induced acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Lonicerae japonicae flos (LJF, the dried flower bud or newly bloomed flower of Lonicera japonica Thunb.), a typical herbal medicine, targets the lung, heart and stomach meridian with the function of clearing heat and detoxication. It ameliorated inflammatory responses and protected against acute lung inflammation in animal models. Acute lung injury (ALI) is a kind of inflammatory disease in which alveolar cells are damaged. However, a network pharmacology study to thoroughly investigate the mechanisms preventing ALI has not been performed. AIM OF THE STUDY: In this study, we examined the main active ingredients in LJF and the protective effects of LJF on LPS-induced ALI in rats. MATERIALS AND METHODS: First, the main active ingredients of LJF were screened in the TCMSP database, and the ALI-associated targets were collected from the GeneCards database. Then, we used compound-target and target-pathway networks to uncover the preventive mechanisms of LJF. Furthermore, we assessed the preventive effects of LJF in an LPS-induced rat model with the RNA-Seq technique to validate the possible molecular mechanisms of the effects of LJF in the treatment of ALI. RESULTS: The network pharmacology results identified 28 main active compounds in LJF, and eight chemical components highly related to the potential targets, which were potential active compounds in LJF. In all, 94 potential targets were recognized, including IL6, TNF, PTGS2, APP, F2, and GRM5. The pathways revealed that the possible targets of LJF involved in the regulation of the IL-17 signalling pathway. Then, in vivo experiments indicated that LJF decreased the levels of proinflammatory cytokines (TNF-, IL-1, and IL-6) in serum and bronchoalveolar lavage fluid, decreased the levels of oxidative stress factors (MDA and MPO) and increased the activities of SOD and GSH-Px in lung tissue. The RNA-Seq results revealed that 7811, 775 and 3654 differentially expressed genes (DEGs) in Ctrl (control group), ALI-LJF (Lonicerae japonicae flos group) and ALI-DXSM (dexamethasone group), respectively. KEGG pathway analysis showed that the DEGs associated with immune response and inflammation signalling pathways and the IL-17 signalling pathway were significantly enriched in LJF. Compared with those in ALI, the expression of CXCL2, CXCL1, CXCL6, NFKBIA, IFNG, IL6, IL17A, IL17F, IL17C, MMP9 and TNFAIP3, which are involved in the IL-17 signalling pathway, were significantly decreased in the LJF group according to the qRT-PCR analyses. CONCLUSIONS: In view of the network pharmacology and RNA-Seq results, the study identified the main active ingredient and potential targets of LJF involved in protecting against ALI, which suggests directions for further research on LJF.