Lung Tissues Of Mice Research Articles

Shuangdan Jiedu Decoction improved LPS-induced acute lung injury by regulating both cGAS-STING pathway and inflammasome

Ethnopharmacological relevanceShuangdan Jiedu Decoction (SJD) is a formula composed of six Chinese herbs with heat-removing and detoxifying, antibacterial, and anti-inflammatory effects, which is clinically used in the therapy of various inflammatory diseases of the lungs including COVID-19, but the therapeutic material basis of its action as well as its molecular mechanism are still unclear. Aim of the studyThe study attempted to determine the therapeutic effect of SJD on LPS-induced acute lung injury (ALI), as well as to investigate its mechanism of action and assess its therapeutic potential for the cure of inflammation-related diseases in the clinical setting. Materials and methodsWe established an ALI model by tracheal drip LPS, and after the administration of SJD, we collected the bronchoalveolar lavage fluid (BALF) and lung tissues of mice and examined the expression of inflammatory factors in them. In addition, we evaluated the effects of SJD on the cyclic guanosine monophosphate-adenosine monophosphate synthase –stimulator of interferon genes (cGAS-STING) and inflammasome by immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR). ResultsWe demonstrated that SJD was effective in alleviating LPS-induced ALI by suppressing the levels of pro-inflammatory cytokines in the BALF, improving the level of lung histopathology and the number of neutrophils, as well as decreasing the inflammatory factor-associated gene expression. Importantly, we found that SJD could inhibit multiple stimulus-driven activation of cGAS-STING and inflammasome. Further studies showed that the Chinese herbal medicines in SJD had no influence on the cGAS-STING pathway and inflammasome alone at the formulated dose. By increasing the concentration of these herbs, we observed inhibitory effects on the cGAS-STING pathway and inflammasome, and the effect exerted was maximal when the six herbs were combined, indicating that the synergistic effects among these herbs plays a crucial role in the anti-inflammatory effects of SJD. ConclusionsOur research demonstrated that SJD has a favorable protective effect against ALI, and its mechanism of effect may be associated with the synergistic effect exerted between six Chinese medicines to inhibit the cGAS-STING and inflammasome abnormal activation. These results are favorable for the wide application of SJD in the clinic as well as for the development of drugs for ALI from herbal formulas.

Dehydrocostus Lactone Ameliorates LPS-Induced Acute Lung Injury by Inhibiting PFKFB3-Mediated Glycolysis.

Acute lung injury (ALI) is a destructive respiratory disease characterized by alveolar structural destruction and excessive inflammation responses. Aerobic glycolysis of macrophages plays a crucial role in the pathophysiology of ALI. Previous studies have shown that the expression of the key rate-limiting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in inflammatory cells is significantly increased, which promotes an increase in the rate of glycolysis in inflammatory cells. However, little is known about the biological functions of PFKFB3 in macrophage inflammation and ALI. In this study, we identified that PFKFB3 is markedly increased in lipopolysaccharide (LPS)-induced ALI mice and macrophages. Knockdown of pfkfb3 attenuated LPS-induced glycolytic flux, decreased the release of pro-inflammatory cytokines, and inactivated NF-κB signaling pathway in macrophages. Subsequently, we found that dehydrocostus lactone (DL), a natural sesquiterpene lactone, significantly decreased both the mRNA and protein levels of PFKFB3. Furthermore, it reduced the release of inflammatory cytokines and inactivated NF-κB pathways in vitro. Accordingly, DL alleviated LPS-induced pulmonary edema and reduced the infiltration of inflammatory cells in mouse lung tissue. In summary, our study reveals the vital role of PFKFB3 in LPS-induced inflammation and discovers a novel molecular mechanism underlying DL's protective effects on ALI.

Local photo-crosslinking of native tissue matrix regulates cell function.

Within most tissues, the extracellular microenvironment provides mechanical cues that guide cell fate and function. Changes in the extracellular matrix such as aberrant deposition, densification and increased crosslinking are hallmarks of late-stage fibrotic diseases that often lead to organ dysfunction. Biomaterials have been widely used to mimic the mechanical properties of the fibrotic matrix and study cell function. However, the initiation of fibrosis has largely been overlooked, due to the challenges in recapitulating early fibrotic lesions within the native extracellular microenvironment. Using visible light mediated photochemistry, we induced local crosslinking and stiffening of extracellular matrix proteins within ex vivo murine and human tissue. In ex vivo lung tissue of epithelial cell lineage-traced mice, local matrix crosslinking mimicked early fibrotic lesions that increased alveolar epithelial cell spreading, differentiation and extracellular matrix remodeling. However, inhibition of cytoskeletal tension or integrin engagement reduced epithelial cell spreading and differentiation, resulting in alveolar epithelial cell dedifferentiation and reduced extracellular matrix deposition. Our findings emphasize the role of local extracellular matrix crosslinking and remodeling in early-stage tissue fibrosis and have implications for ex vivo disease modeling and applications to other tissues.

IL-35 promotes IL-35+IL-10+ Bregs and Conventional LAG3+ Tregs in the lung tissue of OVA-Induced Asthmatic Mice.

This study aimed to investigate the effect of interleukin-35 (IL-35) on inflamed lung tissue in a murine model of asthma. IL-35 was examined for its potential to induce regulatory lymphocytes during ovalbumin (OVA)-induced acute lung injury. Female BALB/c mice sensitized with OVA and were treated with recombinant IL-35 (rIL-35) via intranasal or intraperitoneal routes and were administered 4h before OVA challenge. The effects of rIL-35 treatment on the lung and blood levels of regulatory B cells (Bregs) and regulatory T cells (Tregs), as well as their production of immunosuppressive cytokines, were determined using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Treatment of OVA-sensitized asthmatic mice with rIL-35, whether administered intranasally or intraperitoneally, resulted in reduced lung inflammation and injury. This reduction was accompanied by an increase in the frequency of IL-35 producing Bregs, IL-35 and IL-10 producing Bregs, and conventional LAG3+ Tregs in the lung tissues and blood. This increase was more pronounced with intranasal rIL-35. Furthermore, there was a positive correlation between the levels of these regulatory cells and lung gene expression of IL-35 and IL-10, and an inverse correlation with both lung gene expression and plasma level of IL-17. The results of this study suggest that IL-35, through its ability to increase Bregs and Tregs, is effective in reversing lung inflammation in the context of asthma. Since the increase was more pronounced with intranasal administration, this highlights the therapeutic potential of its local intrapulmonary application in managing asthma-related inflammation.

TNKS1BP1 mediates AECII senescence and radiation induced lung injury through suppressing EEF2 degradation

BackgroundAlthough recent studies provide mechanistic understanding to the pathogenesis of radiation induced lung injury (RILI), rare therapeutics show definitive promise for treating this disease. Type II alveolar epithelial cells (AECII) injury in various manner results in an inflammation response to initiate RILI.ResultsHere, we reported that radiation (IR) up-regulated the TNKS1BP1, causing progressive accumulation of the cellular senescence by up-regulating EEF2 in AECII and lung tissue of RILI mice. Senescent AECII induced Senescence-Associated Secretory Phenotype (SASP), consequently activating fibroblasts and macrophages to promote RILI development. In response to IR, elevated TNKS1BP1 interacted with and decreased CNOT4 to suppress EEF2 degradation. Ectopic expression of EEF2 accelerated AECII senescence. Using a model system of TNKS1BP1 knockout (KO) mice, we demonstrated that TNKS1BP1 KO prevents IR-induced lung tissue senescence and RILI.ConclusionsNotably, this study suggested that a regulatory mechanism of the TNKS1BP1/CNOT4/EEF2 axis in AECII senescence may be a potential strategy for RILI.

An integrative pharmacology-based study on the efficacy and mechanism of essential oil of Chaihu Guizhi Decoction on influenza A virus induced pneumonia in mice

Ethnopharmacological relevanceChaihu Guizhi Decoction (CGD) has a long history of use in China for the treatment of influenza, which involves the use of a variety of aromatic herbs. Our previous studies have found that the contents of aromatic constituents in CGD affected the efficacy of treatment of influenza-infected mice, suggesting a clue that essential oil from CGD may play a relatively important role in ameliorating influenza induced pneumonia. Aim of the studyTo evaluate the anti-influenza potential of essential oil derived from Chaihu Guizhi Decoction (CGD-EO), to characterize and predict the key active components in CGD-EO, and to explore the mechanism of action of CGD-EO. Materials and methodsCGD-EO was obtained by steam distillation, and the components of the essential oil were characterized by gas chromatography-mass spectrometry (GC-MS) in conjunction with the retention index. The constituents absorbed into the blood of mice treated with CGD-EO were analyzed by headspace solid phase microextraction gas chromatography/mass spectrometry (HS-SPME-GC/MS). The potential anti-influenza active constituents and their possible action pathway were predicted by simulation using a network pharmacology approach. The protective effect of CGD-EO and its major components on H1N1/PR8-infected cells was determined using the CCK8 assay kit. Mice infected with influenza A virus H1N1/PR8 were administered different doses of CGD-EO orally and the body weights and lung weights were recorded. Mice with varying degrees of H1N1/PR8 infection were administered CGD-EO orally, and their daily weight, water consumption, and clinical indicators were recorded. Necropsies were conducted on days 3 and 5, during which lung weights were measured and lung tissues were preserved. Furthermore, the mRNA expression of the H1N1/PR8 virus and inflammatory factors in lung tissue was analyzed using RT-qPCR. Results(E)-cinnamaldehyde was the most abundant compound in the CGD-EO. The results of serum medicinal chemistry combined with network pharmacological analysis indicated that (E)-cinnamaldehyde and 3-phenyl-2-propenal may be potential active components of the CGD-EO anti-influenza, and may be involved in the NF-κB signalling pathway. In vitro studies have demonstrated that both CGD-EO and cinnamaldehyde exert a protective effect on MDCK cells infected with H1N1/PR8. In a 0.5 TCID50 H1N1/PR8-induced influenza model, mice treated with CGD-EO at a dose of 63.50 μg/kg exhibited a reduction in lung index, pathological lung lesions, and H1N1/PR8 viral gene levels. In addition, CGD-EO treatment was found to regulate the levels of inflammatory cytokines, including IL-6, TNF-α, and IFN-γ. Moreover, following three days of administration, an upregulation of NF-κB mRNA levels in mouse lung tissue was observed in response to CGD-EO treatment. ConclusionsThe findings of our study indicate CGD-EO exerts a protective effect against H1N1-induced cytopathic lesions in vitro and is capable of alleviating H1N1-induced pneumonitis in mice. Moreover, it appears to be more efficacious in the treatment of mild symptoms of H1N1 infection. Studies have demonstrated that CGD-EO has antiviral potential to attenuate influenza-induced lung injury by modulating inflammatory cytokines and NF-κB signalling pathways during the early stages of influenza infection. It is possible that (E)-cinnamaldehyde is a potential active ingredient in the anti-influenza efficacy of CGD-EO.

High-throughput gene expression analysis with TempO-LINC sensitively resolves complex brain, lung and kidney heterogeneity at single-cell resolution.

We report the development and performance of a novel genomics platform, TempO-LINC, for conducting high-throughput transcriptomic analysis on single cells and nuclei. TempO-LINC works by adding cell-identifying molecular barcodes onto highly selective and high-sensitivity gene expression probes within fixed cells, without having to first generate cDNA. Using an instrument-free combinatorial-indexing approach, all probes within the same fixed cell receive an identical barcode, enabling the reconstruction of single-cell gene expression profiles across as few as several hundred cells and up to 100,000+ cells per run. The TempO-LINC approach is easily scalable based on the number of barcodes and rounds of barcoding performed; however, for the experiments reported in this study, the assay utilized over 5.3 million unique barcodes. TempO-LINC has a robust protocol for fixing and banking cells and displays high-sensitivity gene detection from multiple diverse sample types. We show that TempO-LINC has an observed multiplet rate of less than 1.1% and a cell capture rate of ~50%. Although the assay can accurately profile the whole transcriptome (19,683 human or 21,400 mouse genes), it can be targeted to measure only actionable/informative genes and molecular pathways of interest - thereby reducing sequencing requirements. In this study, we applied TempO-LINC to profile the transcriptomes of 89,722 cells across multiple sample types, including nuclei from mouse lung, kidney and brain tissues. The data demonstrated the ability to identify and annotate at least 50 unique cell populations and positively correlate expression of cell type-specific molecular markers within them. TempO-LINC is a robust new single-cell technology that is ideal for large-scale applications/studies across thousands of samples with high data quality.

Growth Differentiation Factor 11 Evokes Lung Injury, Inflammation, and Fibrosis in Mice through the Activin A Receptor Type II-Like Kinase, 53kDa–Smad2/3 Signaling Pathway

Growth differentiation factor 11 (GDF11) belongs to the transforming growth factor-β superfamily and participates in various pathophysiological processes. Initially, GDF11 was suggested to act as a rejuvenator by improving age-related phenotypes of the heart, brain, and skeletal muscle in aged mice. However, recent studies demonstrate that GDF11 also serves as an adverse risk factor for human frailty and diseases. However, the role of GDF11 in pulmonary fibrosis (PF) remains unclear. In this study, we explored the role and signaling mechanisms of GDF11 in PF. We discovered that GDF11 expression was markedly up-regulated in fibrotic lung tissues of both humans and mice. Intratracheal administration of commercial recombinant GDF11 caused lung injury, inflammation, and fibrogenesis in mice. Furthermore, adenovirus-mediated secretory expression of mature GDF11 was exacerbated, whereas full-length GDF11 or the GDF11 propeptide (GDF111-298) alleviated bleomycin-induced PF in mice. Invitro experiments demonstrated that GDF11 suppressed the growth of alveolar and bronchial epithelial cells (A549 and BEAS-2B) and human pulmonary microvascular endothelial cells, promoted fibroblast activation, and induced epithelial/endothelial-mesenchymal transition. These effects corresponded to the phosphorylation of Smad2/3, and blocking ALK5-Smad2/3 signaling abolished the invivo and invitro effects of GDF11. In conclusion, our findings provide evidence that GDF11 acts as a potent injurious, proinflammatory, and profibrotic factor in the lungs via the ALK5-Smad2/3 pathway.

Nobiletin alleviates macrophage M2 polarization by activating AMPK-mTOR-mediated autophagy in pulmonary fibrosis mice

Nobiletin (NOB), a polymethoxylated flavone isolated from citrus peels, is a promising dietary treatment for lung diseases, such as pulmonary fiborsis. In this work, the underlying mechanisms of NOB’s preventative effect on pulmonary fibrosis were explored using bleomycin-exposed mice and IL-4-induced M2 polarization of the macrophages. Results showed that NOB treatment could significantly ameliorate lung fibrosis by suppressing pathological damages, collagen deposition, and fibroblat activation. Moreover, NOB obviously reduced the M2 macrophage-related proteins, including CD206, Arg1, and pro-fibrotic mediators such as TGF-β and CTGF, which might contribute to the antifibrosis effect of NOB. Network analysis of the differentially expressed genes in NOB-treated M2 macrophages showed that autophagy, mTOR signaling pathway, and AMPK signaling pathway might be involved in the effects of NOB. Further exploration illustrated that autophagy was enhanced in NOB-treated lung and M2 macrophages.The addition of 3MA, an autophagy inhibitor, could significantly weaken the effect of NOB on lung fibrosis and macrophage M2 polarization. Additionally, NOB also markedly decreased the expression of p-AMPK, p-mTOR, and p-P70S6K in the M2 macrophages and lung tissues of BLM-exposed mice. Compound C, an AMPK agonist, significantly suppressed NOB-induced activation of AMPK and mTOR signals, as well as its inhibitory effect on autophagy, M2 macrophages and lung fibrosis both in vitro and in vivo, supporting the requirement of AMPK-mTOR-mediated autophagy for the NOB’s antifibrosis activity. Taken together, this study suggests that NOB ameliorates pulmonary fibrosis likely involving the inhibition of M2 macrophage via activating AMPK-mTOR-mediated autophagy.

Curcumin analogue EF24 prevents alveolar epithelial cell senescence to ameliorate idiopathic pulmonary fibrosis via activation of PTEN

BackgroundTreating Idiopathic pulmonary fibrosis (IPF) remains challenging owing to its relentless progression, grim prognosis, and the scarcity of effective treatment options. Emerging evidence strongly supports the critical role of accelerated senescence in alveolar epithelial cells (AECs) in driving the progression of IPF. Consequently, targeting senescent AECs emerges as a promising therapeutic strategy for IPF. PurposeCurcumin analogue EF24 is a derivative of curcumin and shows heightened bioactivity encompassing anti-inflammatory, anti-tumor and anti-aging properties. The objective of this study was to elucidate the therapeutic potential and underlying molecular mechanisms of EF24 in the treatment of IPF. MethodsA549 and ATII cells were induced to become senescent using bleomycin. Senescence markers were examined using different methods including senescence-associated β-galactosidase (SA-β-gal) staining, western blotting, and q-PCR. Mice were intratracheally administrated with bleomycin to induce pulmonary fibrosis. This was validated by micro-computed tomography (CT), masson trichrome staining, and transmission electron microscope (TEM). The role and underlying mechanisms of EF24 in IPF were determined in vitro and in vivo by evaluating the expressions of PTEN, AKT/mTOR/NF-κB signaling pathway, and mitophagy using western blotting or flow cytometry. ResultsWe identified that the curcumin analogue EF24 was the most promising candidate among 12 compounds against IPF. EF24 treatment significantly reduced senescence biomarkers in bleomycin-induced senescent AECs, including SA-β-Gal, PAI-1, P21, and the senescence-associated secretory phenotype (SASP). EF24 also effectively inhibited fibroblast activation which was induced by senescent AECs or TGF-β. We revealed that PTEN activation was integral for EF24 to inhibit AECs senescence by suppressing the AKT/mTOR/NF-κB signaling pathway. Additionally, EF24 improved mitochondrial dysfunction through induction of mitophagy. Furthermore, EF24 administration significantly reduced the senescent phenotype induced by bleomycin in the lung tissues of mice. Notably, EF24 mitigates fibrosis and promotes overall health benefits in both the acute and chronic phases of IPF, suggesting its therapeutic potential in IPF treatment. ConclusionThese findings collectively highlight EF24 as a new and effective therapeutic agent against IPF by inhibiting senescence in AECs.

Melatonin as a radioprotective agent against flattening filter and flattening filter-free beam in radiotherapy-induced lung tissue damage

Background Radiotherapy is a widely used treatment method in oncology, applied by delivering high-energy particles or waves to the tumor tissue. Although tumor cells are targeted with radiotherapy, it can cause acute or long-term damage to healthy tissues. Therefore, the preservation of healthy tissues has been an important subject of various scientific researches. Melatonin has been shown to have a radioprotective effect on many tissues and organs such as liver, parotid gland, brain, and testicles. This study aimed to evaluate the protective effect of melatonin against the radiation at various doses and rates administered to the lung tissue of healthy mice. Methods This study was a randomized case-control study conducted with 80 rats comprising 10 groups with eight animals per group. Of the 10 groups, first is the control group, which is not given any melatonin, and second is the group that does not receive RT, which is given only melatonin, and the other eight groups are RT groups, four with melatonin and four without melatonin. Results There was no statistical difference in terms of histopathological findings in the lung tissue between the second group, which did not receive radiotherapy and received only melatonin, and the control group. Lung damage due to radiotherapy was statistically significantly higher in the groups that did not receive melatonin compared to the groups that received melatonin. Conclusions This study revealed that melatonin has a protective effect against the cytotoxic damage of RT in rats receiving RT.

CircLMBR1 inhibits phenotypic transformation of hypoxia-induced pulmonary artery smooth muscle via the splicing factor PUF60

Phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) contributes to vascular remodeling in hypoxic pulmonary hypertension (PH). Recent studies have suggested that circular RNAs (circRNAs) may play important roles in the vascular remodeling of hypoxia-induced PH. However, whether circRNAs cause pulmonary vascular remodeling by regulating the phenotypic transformation in PH has not been investigated. Microarray and RT-qPCR analysis identified that circLMBR1, a novel circRNA, decreased in mouse lung tissues of the hypoxia-SU5416 PH model, as well as in human PASMCs and mouse PASMCs exposed to hypoxia. Overexpression of circLMBR1 in the Semaxinib (SU5416) mouse model ameliorated hypoxia-induced PH and vascular remodeling in the lungs. Notably, circLMBR1 was mainly distributed in the nucleus and bound to the splicing factor PUF60. CircLMBR1 suppressed the phenotypic transformation of human PASMCs and vascular remodeling by inhibiting PUF60 expression. Furthermore, we identified U2AF65 as the downstream regulatory factor of PUF60. U2AF65 directly interacted with the pre-mRNA of the contractile phenotype marker smooth muscle protein 22-α (SM22α) and inhibited its splicing. Meanwhile, hypoxia exposure increased the formation of the PUF60–U2AF65 complex, thereby inhibiting SM22α production and inducing the transition of human PASMCs from a contractile phenotype to a synthetic phenotype. Overall, our results verified the important role of circLMBR1 in the pathological process of PH. We also proposed a new circLMBR1/PUF60–U2AF65/pre-SM22α pathway that could regulate the phenotypic transformation and proliferation of human PASMCs. This study may provide new perspectives for the diagnosis and treatment of PH.

Ficus pumila fruit polysaccharide attenuate ovalbumin-induced allergic asthma in mice associated with changes in microbiota involving the lung-intestinal axis

Allergic asthma accounts for more than 50 percent of adult asthma in life. Glucocorticoids and leukotriene receptor antagonists are commonly used to treat asthma, but these drugs not only have high side effects but also cannot cure the disease. In contrast, natural active ingredients are relatively safe to the organism and show great potential for improving the organism. Polysaccharides, as the main active ingredient in aqueous extract, may be equally beneficial for biological activity. The aim of this study is to investigate the alleviating effect and mechanism of action of Ficus pumila fruit polysaccharide (FPP) and aqueous extract of Ficus pumila fruit (FPW) on mice with allergic asthma. The results showed that FPP and FPW could regulate the secretion of the cytokines IL-4, IL-5, IL-13, and IFN-γ. FPP and FPW also reduced the inflammation, mucus secretion, and collagen deposition in mouse lung tissue, and down-regulated the expression of matrix metallopeptidase-9 (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and α-smooth muscle actin (α-SMA) proteins. In addition, FPP and FPW modulated the intestinal microbiota by augmenting the alpha diversity and adjusting the taxonomic composition at the phylum and family levels, thereby ensured the sustenance of regular physiological processes in the body. This study demonstrated that FPP and FPW may attenuate asthma by inducing beneficial microbiota in the gut-lung axis and provide an effective basis for the future clinical treatment of allergic asthma with Ficus pumila and the development and utilization of new anti-asthma drugs.

Ameliorative effect of pedunculoside on sepsis-induced acute lung injury, inflammation and pulmonary fibrosis in mice model via suppressing AKT/NF-κB pathway.

Sepsis-induced acute lung injury (ALI) is the typical complications of sepsis with a high global incidence and mortality. Inhibition of inflammatory response is a crucial and effective strategy for sepsis-induced ALI. Pedunculoside (PE) has been shown to have an anti-inflammatory effect on various diseases. However, the effect and mechanism of PE on sepsis-induced ALI remain unknown. A mice model of sepsis-induced ALI was constructed by cecal ligation and puncture (CLP). The effect of PE on the CLP-induced mice were assessed using pathological staining, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), reverse transcription quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot assays. PE reduced pathological symptoms and scores, apoptosis and the W/D ratio of lung tissues in CLP-induced mice. Besides, PE decreased the level of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α), pulmonary fibrosis and the expression of fibrosis markers. Mechanically, PE inhibited AKT/NF-κB signaling in CLP-induced mice. Activation of AKT/NF-κB pathway abolished the ameliorative effect of PE on the pathological symptoms, the release of inflammatory factors and pulmonary fibrosis of CLP-induced mice. PE improved inflammation and pulmonary fibrosis by inhibiting AKT/NF-κB pathway in CLP-induced mice.

Integration of microbiomics, metabolomics, and transcriptomics reveals the therapeutic mechanism underlying Fuzheng-Qushi decoction for the treatment of lipopolysaccharide-induced lung injury in mice

Ethnopharmacological relevanceFuzheng-Qushi decoction (FZQS) is a practical Chinese herbal formula for relieving cough and fever. Therefore, the action and specific molecular mechanism of FZQS in the treatment of lung injury with cough and fever as the main symptoms need to be further investigated. Aims of the studyTo elucidate the protective effects of FZQS against lung injury in mice and reveal its potential targets and key biological pathways for the treatment of lung injury based on transcriptomics, microbiomics, and untargeted metabolomics analyses. Materials and methodsLipopolysaccharide (LPS) was used to induce a mouse model of lung injury, followed by the administration of FZQS. ELISA was used to detect IL-1β, IL-6, IL-17A, IL-4, IL-10, and TNF-α, in mouse lung tissues. Macrophage polarization and neutrophil activation were measured by flow cytometry. RNA sequencing (RNA-seq) was applied to screen for differentially expressed genes (DEGs) in lung tissues. RT-qPCR and Western blot assays were utilized to validate key DEGs and target proteins in lung tissues. 16S rRNA sequencing was employed to characterize the gut microbiota of mice. Metabolites in the gut were analyzed using untargeted metabolomics. ResultsFZQS treatment significantly ameliorated lung histopathological damage, decreased pro-inflammatory cytokine levels, and increased anti-inflammatory cytokine levels. M1 macrophage levels in the peripheral blood decreased, M2 macrophage levels increased, and activated neutrophils were inhibited in mice with LPS-induced lung injury. Importantly, transcriptomic analysis showed that FZQS downregulated macrophage and neutrophil activation and migration and adhesion pathways by reversing 51 DEGs, which was further confirmed by RT-qPCR and Western blot analysis. In addition, FZQS modulated the dysbiosis of the gut microbiota by reversing the abundance of Corynebacterium, Facklamia, Staphylococcus, Paenalcaligenes, Lachnoclostridium, norank_f_Muribaculaceae, and unclassified_f_Lachnospiraceae. Meanwhile, metabolomics analysis revealed that FZQS significantly regulated tryptophan metabolism by reducing the levels of 3-Indoleacetonitrile and 5-Hydroxykynurenine. ConclusionFZQS effectively ameliorated LPS-induced lung injury by inhibiting the activation, migration, and adhesion of macrophages and neutrophils and modulating gut microbiota and its metabolites.

Sodium houttuyfonate Modulates the Lung Th1/Th2 Balance and Gut Microbiota to Protect Against Pathological Changes in Lung of Ovalbumin-induced Asthmatic mice

Objective The gut-lung axis involves microbial and product interactions between the lung and intestine. Antibiotics for chronic asthma can cause intestinal dysbiosis, disrupting this axis. Sodium houttuyfonate (SH) has diverse biological activities, including modifying gut microbiota, antibacterial, and anti-inflammatory. This study aims to explore the relationship between SH, CD4+ T cells, and gut microbiota. Methods Allergic asthma was experimentally induced in mice through injection and inhalation of ovalbumin. After the administration of different amounts of SH, ELISA was utilized to ascertain the levels of inflammatory cytokines in the serum, flow cytometry was used to examine the levels of Th1/Th2 cytokines in CD4+ cells from lung tissues. The expression of T-bet and GATA3 in lung tissue was determined by Western blotting and quantitative real-time PCR assay. Gut microbiota was determined by 16S rRNA gene sequencing. Results The results showed that SH can alleviate pulmonary injury in asthmatic mice, reducing serum levels of IL-4, IL-5, and IL-13 while simultaneously increasing IFN-γ. Furthermore, SH has been observed to modulate the balance of Th1/Th2 cells by up-regulating the mRNA and protein expression of T-bet but down-regulating GATA3 in the lung tissues of asthmatic mice, thereby promoting the differentiation of Th1 cells. Additionally, SH can regulate the variety and composition of gut microbiota especially genus Akkermansia in asthmatic mice. Conclusion SH can alleviate asthma through the regulation of Th1/Th2 cells and gut microbiota.

METTL3-m6A methylation inhibits the proliferation and viability of type II alveolar epithelial cells in acute lung injury by enhancing the stability and translation efficiency of Pten mRNA

BackgroundThe pathogenesis of acute lung injury (ALI) involves a severe inflammatory response, leading to significant morbidity and mortality. N6-methylation of adenosine (m6A), an abundant mRNA nucleotide modification, plays a crucial role in regulating mRNA metabolism and function. However, the precise impact of m6A modifications on the progression of ALI remains elusive.MethodsALI models were induced by either intraperitoneal injection of lipopolysaccharide (LPS) into C57BL/6 mice or the LPS-treated alveolar type II epithelial cells (AECII) in vitro. The viability and proliferation of AECII were assessed using CCK-8 and EdU assays. The whole-body plethysmography was used to record the general respiratory functions. M6A RNA methylation level of AECII after LPS insults was detected, and then the “writer” of m6A modifications was screened. Afterwards, we successfully identified the targets that underwent m6A methylation mediated by METTL3, a methyltransferase-like enzyme. Last, we evaluated the regulatory role of METTL3-medited m6A methylation at phosphatase and tensin homolog (Pten) in ALI, by assessing the proliferation, viability and inflammation of AECII.ResultsLPS induced marked damages in respiratory functions and cellular injuries of AECII. The m6A modification level in mRNA and the expression of METTL3, an m6A methyltransferase, exhibited a notable rise in both lung tissues of ALI mice and cultured AECII cells subjected to LPS treatment. METTL3 knockdown or inhibition improved the viability and proliferation of LPS-treated AECII, and also reduced the m6A modification level. In addition, the stability and translation of Pten mRNA were enhanced by METTL3-mediated m6A modification, and over-expression of PTEN reversed the protective effect of METTL3 knockdown in the LPS-treated AECII.ConclusionsThe progression of ALI can be attributed to the elevated levels of METTL3 in AECII, as it promotes the stability and translation of Pten mRNA through m6A modification. This suggests that targeting METTL3 could offer a novel approach for treating ALI.

The m6A eraser FTO suppresses ferroptosis via mediating ACSL4 in LPS-induced macrophage inflammation

Acute lung injury (ALI) is a serious disorder characterized by the release of pro-inflammatory cytokines and cascade activation of macrophages. Ferroptosis, a form of iron-dependent cell death triggered by intracellular phospholipid peroxidation, has been implicated as an internal mechanism underlying ALI. In this study, we investigated the effects of m6A demethylase fat mass and obesity-associated protein (FTO) on the inhibition of macrophage ferroptosis in ALI. Using a mouse model of lipopolysaccharide (LPS)-induced ALI, we observed the induction of ferroptosis and its co-localization with the macrophage marker F4/80, suggesting that ferroptosis might be induced in macrophages. Ferroptosis was promoted during LPS-induced inflammation in macrophages in vitro, and the inflammation was counteracted by the ferroptosis inhibitor ferrostatin-1 (fer-1). Given that FTO showed lower expression levels in the lung tissue of mice with ALI and inflammatory macrophages, we further dissected the regulatory capacity of FTO in ferroptosis. The results demonstrated that FTO alleviated macrophage inflammation by inhibiting ferroptosis. Mechanistically, FTO decreased the stability of ACSL4 mRNA via YTHDF1, subsequently inhibiting ferroptosis and inflammation by interrupting polyunsaturated fatty acid consumption. Moreover, FTO downregulated the synthesis and secretion of prostaglandin E2, thereby reducing ferroptosis and inflammation. In vivo, the FTO inhibitor FB23–2 aggravated lung injury, the inflammatory response, and ferroptosis in mice with ALI; however, fer-1 therapy mitigated these effects. Overall, our findings revealed that FTO may function as an inhibitor of the inflammatory response driven by ferroptosis, emphasizing its potential as a target for ALI treatment.

Up-Regulation of S100A8 and S100A9 in Pulmonary Immune Response Induced by a Mycoplasma capricolum subsp. capricolum HN-B Strain.

Mycoplasma capricolum subsp. capricolum (Mcc), a member of the Mycoplasma mycoides cluster, has a negative impact on the goat-breeding industry. However, little is known about the pathogenic mechanism of Mcc. This study infected mice using a previously isolated strain, Mcc HN-B. Hematoxylin and eosin staining, RNA sequencing, bioinformatic analyses, RT-qPCR, and immunohistochemistry were performed on mouse lung tissues. The results showed that 235 differentially expressed genes (DEGs) were identified. GO and KEGG enrichment analyses suggested that the DEGs were mainly associated with immune response, defensive response to bacteria, NF-kappa B signaling pathway, natural killer cell-mediated cytotoxicity, and T cell receptor signaling pathway. RT-qPCR verified the expression of Ccl5, Cd4, Cd28, Il2rb, Lck, Lat, Ptgs2, S100a8, S100a9, and Il-33. The up-regulation of S100A8 and S100A9 at the protein level was confirmed by immunohistochemistry. Moreover, RT-qPCR assays on Mcc HN-B-infected RAW264.7 cells also showed that the expression of S100a8 and S100a9 was elevated. S100A8 and S100A9 not only have diagnostic value in Mcc infection but also hold great significance in clarifying the pathogenic mechanism of Mcc. This study preliminarily elucidates the mechanism of Mcc HN-B-induced lung injury and provides a theoretical basis for further research on Mcc-host interactions.

ETS1 and RBPJ transcriptionally regulate METTL14 to suppress TGF-β1-induced epithelial-mesenchymal transition in human bronchial epithelial cells

Asthma is a chronic respiratory disease characterized by airway inflammation and remodeling. Epithelial-mesenchymal transition (EMT) of bronchial epithelial cells is considered to be a crucial player in asthma. Methyltransferase-like 14 (METTL14), an RNA methyltransferase, is implicated in multiple pathological processes, including EMT, cell proliferation and migration. However, the role of METTL14 in asthma remains uncertain. This research aimed to explore the biological functions of METTL14 in asthma and its underlying upstream mechanisms. METTL14 expression was down-regulated in asthmatic from three GEO datasets (GSE104468, GSE165934, and GSE74986). Consistent with this trend, METTL14 was decreased in the lung tissues of OVA-induced asthmatic mice and transforming growth factor-β1 (TGF-β1)-stimulated human bronchial epithelial cells (Beas-2B) in this study. Overexpression of METTL14 caused reduction in mesenchymal markers (FN1, N-cad, Col-1 and α-SMA) in TGF-β1-treated cells, but caused increase in epithelial markers (E-cad), thus inhibiting EMT. Also, METTL14 suppressed the proliferation and migration ability of TGF-β1-treated Beas-2B cells. Two transcription factors, ETS1 and RBPJ, could both bind to the promoter region of METTL14 and drive its expression. Elevating METTL14 expression could reversed EMT, cell proliferation and migration promoted by ETS1 or RBPJ deficiency. These results indicate that the ETS1/METTL14 and RBPJ/METTL14 transcription axes exhibit anti-EMT, anti-proliferation and anti-migration functions in TGF-β1-induced bronchial epithelial cells, implying that METTL14 may be considered an alternative candidate target for the treatment of asthma.