Combination of androgen and estrogen improves asthma by mediating Runx3 expression.

Objective: Asthma is a chronic heterogeneous airway disease, and imbalanced T-helper type 1 (Th1) and Th2 cell-mediated inflammation contribute to its pathogenesis. Although it has been suggested that androgen and estrogen were involved in development of asthma, the underlying mechanisms remained largely unclear. Studies have demonstrated that Runx3 could promote naive CD4+ T cells to differentiate into Th1 cells. Hence, our study aimed to explore the potential regulatory mechanism of androgen and estrogen on asthma via modulating Runx3. Methods: First, clinical assessments and pulmonary function tests were conducted on 35 asthma patients and 24 healthy controls. The concentrations of androgen, estrogen, and androgen estrogen ratios were assessed in peripheral blood samples of asthma patients and healthy controls. Then, a murine asthma model was established to explore the effects of estrogen and androgen (alone or in combination) on asthma. Third, an in vitro assay was used to explore the mechanism of combination of androgen and estrogen in asthma. Results: We observed decreased androgen and increased estrogen levels in asthma patients compared with healthy controls. In mice with experimental asthma, there were increased serum concentrations of estrogen and decreased serum concentrations of androgen, intervention with combination of androgen and estrogen alleviated airway inflammations, increased Runx3 expressions and elevated Th1 differentiation. In CD4+ T cells co-cultured with bronchial epithelial cells (BECs), treatment with androgen plus estrogen combination promoted Th1 differentiation, which was mitigated by Runx3 knockdown in BECs and enhanced by Runx3 overexpression. Conclusion: These findings suggest that androgen estrogen combination modulate the Th1/Th2 balance via regulating the expression of Runx3 in BECs, thereby providing experimental evidence supporting androgen and estrogen combination as a novel therapy for asthma.

A novel tumor mutation-related long non-coding RNA signature for predicting overall survival and immunotherapy response in lung adenocarcinoma.

Background: Immunotherapy has changed the treatment landscape for lung cancer. This study aims to construct a tumor mutation-related model that combines long non-coding RNA (lncRNA) expression levels and tumor mutation levels in tumor genomes to detect the possibilities of the lncRNA signature as an indicator for predicting the prognosis and response to immunotherapy in lung adenocarcinoma (LUAD). Methods: We downloaded the tumor mutation profiles and RNA-seq expression database of LUAD from The Cancer Genome Atlas (TCGA). Differentially expressed lncRNAs were extracted based on the cumulative number of mutations. Cox regression analyses were used to identify the prognostic lncRNA signature, and the prognostic value of the five selected lncRNAs was validated by using survival analysis and the receiver operating characteristic (ROC) curve. We used qPCR to validate the expression of five selected lncRNAs between human lung epithelial and human lung adenocarcinoma cell lines. The ImmuCellAI, immunophenoscore (IPS) scores and Tumor Immune Dysfunction and Exclusion (TIDE) analyses were used to predict the response to immunotherapy for this mutation related lncRNA signature. Results: A total of 162 lncRNAs were detected among the differentially expressed lncRNAs between the Tumor mutational burden (TMB)-high group and the TMB-low group. Then, five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as tumor mutation-related candidates for constructing the prognostic prediction model. Kaplan‒Meier curves showed that the overall survival of the low-risk group was significantly better than that of the high-risk group, and the results of the GSE50081 set were consistent. The expression levels of PD1, PD-L1 and CTLA4 in the low-risk group were higher than those in the high-risk group. The IPS scores and TIDE scores of patients in the low-risk group were significantly higher than those in the high-risk group. Conclusion: Our findings demonstrated that the five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as candidates for constructing the tumor mutation-related model which may serve as an indicator of tumor mutation levels and have important implications for predicting the response to immunotherapy in LUAD.

Hepatocyte-specific METTL3 ablation by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and postnatal lethality.

AIM: In 2019, to examine the functions of METTL3 in liver and underlying mechanisms, we generated mice with hepatocyte-specific METTL3 homozygous knockout (METTL3Δhep) by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT) or Alb-Cre mice (JAX), respectively. In this study, we explored the potential reasons why hepatocyte-specific METTL3 homozygous disruption by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and then postnatal lethality. MAIN METHODS: Mice with hepatocyte-specific METTL3 knockout were generated by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT; Strain No. T003814) purchased from the GemPharmatech Co., Ltd., (Nanjing, China) or with Alb-Cre mice (JAX; Strain No. 003574) obtained from The Jackson Laboratory, followed by combined-phenotype analysis. The publicly available RNA-sequencing data deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession No.: GSE198512 (postnatal lethality), GSE197800 (postnatal survival) and GSE176113 (postnatal survival) were mined to explore the potential reasons why hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), leads to ALF and then postnatal lethality. KEY FINDINGS: Firstly, we observed that hepatocyte-specific METTL3 homozygous deficiency by Alb-iCre mice (GPT) or by Alb-Cre mice (JAX) caused liver injury, abnormal lipid accumulation and apoptosis. Secondly, we are surprised to find that hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), led to ALF and then postnatal lethality. Our findings clearly demonstrated that METTL3Δhep mice (GPT), which are about to die, exhibited the severe destruction of liver histological structure, suggesting that METTL3Δhep mice (GPT) nearly lose normal liver function, which subsequently contributes to ALF, followed by postnatal lethality. Finally, we unexpectedly found that as the compensatory growth responses of hepatocytes to liver injury induced by METTL3Δhep (GPT), the proliferation of METTL3Δhep hepatocytes (GPT), unlike METTL3Δhep hepatocytes (JAX), was not evidenced by the significant increase of Ki67-positive hepatocytes, not accompanied by upregulation of cell-cycle-related genes. Moreover, GO analysis revealed that upregulated genes in METTL3Δhep livers (GPT), unlike METTL3Δhep livers (JAX), are not functionally enriched in terms associated with cell cycle, cell division, mitosis, microtubule cytoskeleton organization, spindle organization, chromatin segregation and organization, and nuclear division, consistent with the loss of compensatory proliferation of METTL3Δhep hepatocytes (GPT) observed in vivo. Thus, obviously, the loss of the compensatory growth capacity of METTL3Δhep hepatocytes (GPT) in response to liver injury might contribute to, at least partially, ALF and subsequently postnatal lethality of METTL3Δhep mice (GPT). SIGNIFICANCE: These findings from this study and other labs provide strong evidence that these phenotypes (i.e., ALF and postnatal lethality) of METTL3Δhep mice (GPT) might be not the real functions of METTL3, and closely related with Alb-iCre mice (GPT), suggesting that we should remind researchers to use Alb-iCre mice (GPT) with caution to knockout gene in hepatocytes in vivo.

METTL3 mediates SOX5 m6A methylation in bronchial epithelial cells to attenuate Th2 cell differentiation in T2 asthma.

Objective: Asthma, a chronic inflammatory disease in which type 2 T helper cells (Th2) play a causative role in the development of T2 asthma. N6-methyladenosine (m6A) modification, an mRNA modification, and methyltransferase-like 3 (METTL3) is involved in the development of T2 asthma by inhibiting Th2 cell differentiation. Sex determining region Y-box protein 5 (SOX5) is involved in regulating T cell differentiation, but its role in T2 asthma was unclear. The objective of this study was to explore the role of METTL3 and SOX5 in T2 asthma and whether there is an interaction between the two. Materials and methods: Adults diagnosed with T2 asthma (n = 14) underwent clinical information collection and pulmonary function tests. In vivo and in vitro T2 asthma models were established using female C57BL/6 mice and human bronchial epithelial cells (HBE). The expressions of METTL3 and SOX5 were detected by Western blot and qRT-PCR and Western blot. Th2 cell differentiation was determined by flow cytometry and IL-4 level was detected by ELISA. m6A methylation level was determined by m6A quantitative assay. The relationship between METTL3 expression and clinical parameters was determined by Spearman rank correlation analysis. The function of METTL3 and SOX5 genes in asthma was investigated in vitro and in vivo. The RNA immunoprecipitation assay detected the specific interaction between METTL3 and SOX5. Results: Patients with T2 asthma displayed lower METTL3 levels compared to healthy controls. Within this group, a negative correlation was observed between METTL3 and Th2 cells, while a positive correlation was noted between METTL3 and clinical parameters as well as Th1 cells. In both in vitro and in vivo models representing T2 asthma, METTL3 levels decreased significantly, while SOX5 levels showed the opposite trend. Overexpression of METTL3 gene in HBE cells significantly inhibited Th2 cell differentiation and increased m6A methylation activity. From a mechanism perspective, low METTL3 negatively regulates SOX5 expression through m6A modification dependence, while high SOX5 expression is positively associated with T2 asthma severity. Cell transfection experiments confirmed that METTL3 regulates Th2 cell differentiation and IL-4 release through SOX5. Conclusions: Overall, our results indicate that METTL3 alleviates Th2 cell differentiation in T2 asthma by modulating the m6A methylation activity of SOX5 in bronchial epithelial cells. This mechanism could potentially serve as a target for the prevention and management of T2 asthma.

Serplulimab monotherapy in extensive-stage small-cell lung cancer with brain metastasis: a case report.

Small-cell lung cancer (SCLC) is an aggressive form of lung cancer with limited treatment options, especially for extensive-stage (ES) patients. We present a case of a 70-year-old male with ES-SCLC and asymptomatic brain metastasis who opted for immune monotherapy with serplulimab (an anti-PD-1 antibody). After four cycles, the patient achieved a confirmed partial response and a progression-free survival of over 1 year. Moreover, we observed a consistent decline in tumor biomarkers, and brain MRI indicated reduced metastatic activity. Remarkably, the patient tolerated the treatment well, with only mild diarrhea. This case highlights serplulimab's potential as a first-line treatment in select ES-SCLC patients, emphasizing the importance of further research on immunotherapy predictive biomarkers.

Small-cell lung cancer (SCLC) is a severe type of lung cancer that often does not have many treatment options, especially in its advanced stages. This article discusses the experience of a 70-year-old man with advanced SCLC who also had cancer spread to his brain but did not show symptoms. He chose to try a new kind of cancer treatment called serplulimab, which works by helping the immune system fight the cancer. After receiving this treatment four-times, his cancer showed significant improvement, and he did not experience further cancer growth for more than 1 year. Tests also revealed that his cancer markers decreased, and the cancer in his brain became less active. Notably, he tolerated this agent with only mild diarrhea occurring. This case is important because it suggests that serplulimab could be an effective first treatment for some patients with advanced SCLC, and it highlights the need for more research to find ways to predict who will benefit from this type of therapy.

circRNA-CPA4 Regulates Cell Proliferation and Apoptosis of Non-small Cell Lung Cancer via the miR-1183/PDPK1 Axis.

Non-small-cell lung cancer (NSCLC) stands as a prevalent subtype of lung cancer, with circular RNAs emerging as key players in cancer development. This study elucidates the role of circRNA-CPA4 in NSCLC. Elevated circRNA-CPA4 expression in NSCLC lines was confirmed through qRT-PCR. Silencing circRNA-CPA4 with shRNA revealed, through CCK-8, colony formation, and flow cytometry assays, a notable constraint on proliferation and promotion of apoptosis in NSCLC cells. Subcellular localization analysis, RNA immunoprecipitation, and expression level assessments were employed to decipher the intricate interplay among miR-1183, circRNA-CPA4, and PDPK1. Results demonstrated heightened circRNA-CPA4 levels in NSCLC, and its knockdown curtailed NSCLC growth in vivo. Acting as a molecular sponge for miR-1183, circRNA-CPA4 regulated PDPK1 expression. Conversely, inhibiting miR-1183 counteracted the impact of circRNA-CPA4 silencing, reinstating NSCLC cell proliferation, and impeding apoptosis. Overall, this study unveils a novel mechanism: circRNA-CPA4 promotes PDPK1 expression by sequestering miR-1183, fostering NSCLC cell proliferation, and hindering apoptosis.

Obstructive Sleep Apnea Plasma-Derived Exosomes Mediate Cognitive Impairment Through Hippocampal Neuronal Cell Pyroptosis.

OBJECTIVE: Obstructive sleep apnea (OSA) is associated with impaired cognitive function. Exosomes are secreted by most cells and play a role in OSA-associated cognitive impairment (CI). The aim of this study was to investigate whether OSA plasma-derived exosomes cause CI through hippocampal neuronal cell pyroptosis, and to identify exosomal miRNAs in OSA plasma-derived. MATERIALS AND METHODS: Plasma-derived exosomes were isolated from patients with severe OSA and healthy comparisons. Daytime sleepiness and cognitive function were assessed using the Epworth Sleepiness Scale (ESS) and the Beijing version of the Montreal Cognitive Assessment Scale (MoCA). Exosomes were coincubated with mouse hippocampal neurons (HT22) cells to evaluate the effect of exosomes on pyroptosis and inflammation of HT22 cells. Meanwhile, exosomes were injected into C57BL/6 male mice via caudal vein, and then morris water maze was used to evaluate the spatial learning and memory ability of the mice, so as to observe the effects of exosomes on the cognitive function of the mice. Western blot and qRT-PCR were used to detect the expressions of Gasdermin D (GSDMD) and Caspase-1 to evaluate the pyroptosis level. The expression of IL-1ß, IL-6, IL-18 and TNF-α was detected by qRT-PCR to assess the level of inflammation. Correlations of GSDMD and Caspase-1 expression with clinical parameters were evaluated using Spearman's rank correlation analysis. In addition, plasma exosome miRNAs profile was identified, followed by Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. RESULTS: Compared to healthy comparisons, body mass index (BMI), apnea-hypopnea index (AHI), oxygen desaturation index (ODI), and ESS scores were increased in patients with severe OSA, while lowest oxygen saturation during sleep (LSaO2), mean oxygen saturation during sleep (MSaO2) and MoCA scores were decreased. Compared to the PBS group (NC) and the healthy comparison plasma-derived exosomes (NC-EXOS), the levels of GSDMD and Caspase-1 and IL-1ß, IL-6, IL-18 and TNF-α were increased significantly in the severe OSA plasma-derived exosomes (OSA-EXOS) coincubated with HT22 cells. Compared to the NC and NC-EXOS groups, the learning and memory ability of mice injected with OSA-EXOS was decreased, and the expression of GSDMD and Caspase-1 in hippocampus were significantly increased, along with the levels of IL-1ß, IL-6, IL-18 and TNF-α. Spearman correlation analysis found that clinical AHI in HCs and severe OSA patients was positively correlated with GSDMD and Caspase-1 in HT22 cells from NC-EXOS and OSA-EXOS groups, while negatively correlated with clinical MoCA. At the same time, clinical MoCA in HCs and severe OSA patients was negatively correlated with GSDMD and Caspase-1 in HT22 cells from NC-EXOS and OSA-EXOS groups. A unique exosomal miRNAs profile was identified in OSA-EXOS group compared to the NC-EXOS group, in which 28 miRNAs were regulated and several KEGG and GO pathways were identified. CONCLUSIONS: The results of this study show a hypothesis that plasma-derived exosomes from severe OSA patients promote pyroptosis and increased expression of inflammatory factors in vivo and in vitro, and lead to impaired cognitive function in mice, suggesting that OSA-EXOS can mediate CI through pyroptosis of hippocampal neurons. In addition, exosome cargo from OSA-EXOS showed a unique miRNAs profile compared to NC-EXOS, suggesting that plasma exosome associated miRNAs may reflect the differential profile of OSA related diseases, such as CI.

VarCards2: an integrated genetic and clinical database for ACMG-AMP variant-interpretation guidelines in the human whole genome.

VarCards, an online database, combines comprehensive variant- and gene-level annotation data to streamline genetic counselling for coding variants. Recognising the increasing clinical relevance of non-coding variations, there has been an accelerated development of bioinformatics tools dedicated to interpreting non-coding variations, including single-nucleotide variants and copy number variations. Regrettably, most tools remain as either locally installed databases or command-line tools dispersed across diverse online platforms. Such a landscape poses inconveniences and challenges for genetic counsellors seeking to utilise these resources without advanced bioinformatics expertise. Consequently, we developed VarCards2, which incorporates nearly nine billion artificially generated single-nucleotide variants (including those from mitochondrial DNA) and compiles vital annotation information for genetic counselling based on ACMG-AMP variant-interpretation guidelines. These annotations include (I) functional effects; (II) minor allele frequencies; (III) comprehensive function and pathogenicity predictions covering all potential variants, such as non-synonymous substitutions, non-canonical splicing variants, and non-coding variations and (IV) gene-level information. Furthermore, VarCards2 incorporates 368 820 266 documented short insertions and deletions and 2 773 555 documented copy number variations, complemented by their corresponding annotation and prediction tools. In conclusion, VarCards2, by integrating over 150 variant- and gene-level annotation sources, significantly enhances the efficiency of genetic counselling and can be freely accessed at http://www.genemed.tech/varcards2/.

Evaluating the performance of low-frequency variant calling tools for the detection of variants from short-read deep sequencing data.

Detection of low-frequency variants with high accuracy plays an important role in biomedical research and clinical practice. However, it is challenging to do so with next-generation sequencing (NGS) approaches due to the high error rates of NGS. To accurately distinguish low-level true variants from these errors, many statistical variants calling tools for calling low-frequency variants have been proposed, but a systematic performance comparison of these tools has not yet been performed. Here, we evaluated four raw-reads-based variant callers (SiNVICT, outLyzer, Pisces, and LoFreq) and four UMI-based variant callers (DeepSNVMiner, MAGERI, smCounter2, and UMI-VarCal) considering their capability to call single nucleotide variants (SNVs) with allelic frequency as low as 0.025% in deep sequencing data. We analyzed a total of 54 simulated data with various sequencing depths and variant allele frequencies (VAFs), two reference data, and Horizon Tru-Q sample data. The results showed that the UMI-based callers, except smCounter2, outperformed the raw-reads-based callers regarding detection limit. Sequencing depth had almost no effect on the UMI-based callers but significantly influenced on the raw-reads-based callers. Regardless of the sequencing depth, MAGERI showed the fastest analysis, while smCounter2 consistently took the longest to finish the variant calling process. Overall, DeepSNVMiner and UMI-VarCal performed the best with considerably good sensitivity and precision of 88%, 100%, and 84%, 100%, respectively. In conclusion, the UMI-based callers, except smCounter2, outperformed the raw-reads-based callers in terms of sensitivity and precision. We recommend using DeepSNVMiner and UMI-VarCal for low-frequency variant detection. The results provide important information regarding future directions for reliable low-frequency variant detection and algorithm development, which is critical in genetics-based medical research and clinical applications.

Inhibition of METTL3 alleviated LPS-induced alveolar epithelial cell apoptosis and acute lung injury via restoring neprilysin expression.

AIMS: To investigate the role and mechanisms of methyltransferase-like 3 (METTL3) in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury (ALI). MAIN METHODS: LPS intratracheally instillation was applied in alveolar epithelial cell METTL3 conditional knockout (METTL3-CKO) mice and their wild-type littermates. In addition, METTL3 inhibitor STM2457 was used. LPS treatment on mouse lung epithelial 12 (MLE-12) cell was applied to establish an in vitro model of LPS-induced ALI. H&E staining, lung wet-to-dry ratio, and total broncho-alveolar lavage fluid (BALF) concentrations were used to evaluate lung injury. Overall, the m6A level was determined with the m6A RNA Methylation Quantification Kit and dot blot assay. Expression of METTL3 and neprilysin were measured with immunohistochemistry, immunofluorescence, immunofluorescence-fluorescence in situ hybridization, and western blot. Apoptosis was detected with TUNEL, western blot, and flow cytometry. The interaction of METTL3 and neprilysin was determined with RIP-qPCR and MeRIP. KEY FINDINGS: METTL3 expression and apoptosis were increased in alveolar epithelial cells of mice treated with LPS, and METTL3-CKO or METTL3 inhibitor STM2457 could alleviate apoptosis and LPS-induced ALI. In MLE-12 cells, LPS-Induced METTL3 expression and apoptosis. Knockdown of METTL3 alleviated, while overexpression of METTL3 exacerbated LPS-induced apoptosis. LPS treatment reduced neprilysin expression, the intervention of neprilysin expression negatively regulated apoptosis without affecting METTL3 expression, and mitigated the promoting effect of METTL3 on LPS-induced apoptosis. Additionally, METTL3 could bind to the mRNA of neprilysin, and reduce its expression. SIGNIFICANCE: Our findings revealed that inhibition of METTL3 could exert anti-apoptosis and ALI-protective effects via restoring neprilysin expression.

HNRNPA2B1-mediated m6A modification of lncRNA MEG3 facilitates tumorigenesis and metastasis of non-small cell lung cancer by regulating miR-21-5p/PTEN axis.

BACKGROUND: Accumulating data indicate that N6-methyladenosine (m6A) RNA methylation and lncRNA deregulation act crucial roles in cancer progression. Heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) as an m6A "reader" has been reported to be an oncogene in multiple malignancies. We herein aimed to elucidate the role and underlying mechanism by which HNRNPA2B1-mediated m6A modification of lncRNAs contributes to non-small cell lung cancer (NSCLC). METHODS: The expression levels of HNRNPA2B1 and their association with the clinicopathological characteristics and prognosis in NSCLC were determined by RT-qPCR, Western blot, immunohistochemistry and TCGA dataset. Then, the role of HNRNPA2B1 in NSCLC cells was assessed by in vitro functional experiments and in vivo tumorigenesis and lung metastasis models. HNRNPA2B1-mediated m6A modification of lncRNAs was screened by m6A-lncRNA epi-transcriptomic microarray and verified by methylated RNA immunoprecipitation (Me-RIP). The lncRNA MEG3-specific binding with miR-21-5p was evaluated by luciferase gene report and RIP assays. The effects of HNRNPA2B1 and (or) lncRNA MEG3 on miR-21-5p/PTEN/PI3K/AKT signaling were examined by RT-qPCR and Western blot analyses. RESULTS: We found that upregulation of HNRNPA2B1 was associated with distant metastasis and poor survival, representing an independent prognostic factor in patients with NSCLC. Knockdown of HNRNPA2B1 impaired cell proliferation and metastasis in vitro and in vivo, whereas ectopic expression of HNRNPA2B1 possessed the opposite effects. Mechanical investigations revealed that lncRNA MEG3 was an m6A target of HNRNPA2B1 and inhibition of HNRNPA2B1 decreased MEG3 m6A levels but increased its mRNA levels. Furthermore, lncRNA MEG3 could act as a sponge of miR-21-5p to upregulate PTEN and inactivate PI3K/AKT signaling, leading to the suppression of cell proliferation and invasion. Low expression of lncRNA MEG3 or elevated expression of miR-21-5p indicated poor survival in patients with NSCLC. CONCLUSIONS: Our findings uncover that HNRNPA2B1-mediated m6A modification of lncRNA MEG3 promotes tumorigenesis and metastasis of NSCLC cells by regulating miR-21-5p/PTEN axis and may provide a therapeutic target for NSCLC.

Clinical characteristics and risk factors of patients with eosinophilic fasciitis associated with pleural effusion.

To investigate the risk factors of eosinophilic fasciitis (EF) associated with pleural effusion (PE). A retrospective analysis was performed on 22 patients with EF diagnosed by skin biopsy in our hospital, and they were divided into EF-PE and EF according to chest computed tomography examination. The clinical characteristics, clinical manifestations, comorbidities and laboratory test indicators of the two groups were collected and compared, and the risk factors for occurring PE in patients with EF were determined by multivariate logistic regression analysis. Among 22 patients with EF, 8 had PE. The age, course of disease, incidence of fever, weight loss, cough and shortness of breath, pulmonary infection, hypothyroidism, hydronephrosis and kidney stone, swelling rate of small vascular endothelial cells, consolidation shadows, C-reactive protein and thyroid stimulating hormone in EF-PE group were higher than those in EF group, while free triiodothyronine and thyroxine were lower than those in EF group. Age, fever, shortness of breath, C-reactive protein, ESR, thyroid stimulating hormone, pulmonary infection, hypothyroidism, hydronephrosis, kidney stones, swollen small vascular endothelial cells and chest CT consolidation shadows were identified as risk factors for happening PE in patients with EF, while free triiodothyronine and free thyroxine were identified as protective factors against PE in patients with EF. The incidence of EF-PE was 36.36% in this study. Advanced age, high C-reactive protein, ESR, thyroid stimulating hormone, incidence of fever, shortness of breath, pulmonary infection, hydronephrosis, kidney stones, swollen small vascular endothelial cells, chest CT consolidation shadows, and low free triiodothyronine and thyroxine suggest that patients with EF are significantly at increased risk of PE.

miR-146a-3p as a potential novel therapeutic by targeting MBD2 to mediate Th17 differentiation in Th17 predominant neutrophilic severe asthma.

Th17 (T-helper 17) cells subtype of non-T2 (non-type 2) asthma is related to neutrophilic infiltration and resistance to inhaled corticosteroids (ICS), so is also known as severe asthma. Methyl-CpG binding domain protein 2 (MBD2) regulates the differentiation of the Th17 cells, tending to show a therapeutic target in severe asthma. miR-146a-3p is associated with anti-inflammatory characteristics and immunity. Moreover, bioinformatic analysis showed that MBD2 may be a target gene of miR-146a-3p. However, the role of miR-146a-3p in the differentiation of Th17 cells via MBD2 in severe asthma remains unknown. Here, we aimed to explore how miR-146a-3p interacts with MBD2 and affects the differentiation of Th17 cells in severe asthma. First, we recruited 30 eligible healthy people and 30 patients with severe asthma to detect the expression of miR-146a-3p in peripheral blood mononuclear cells (PBMCs) by qRT-PCR. Then, we established a HDM/LPS (house dust mite/lipopolysaccharide) exposure model of bronchial epithelial cells (BECs) to evaluate the expression of miR-146a-3p, the interaction between miR-146a-3p and MBD2 using western blot and luciferase reporter analysis and the effect of miR-146a-3p regulated Th17 cells differentiation by flow cytometry in BECs in vitro. Finally, we constructed a mouse model of Th17 predominant neutrophilic severe asthma to assess the therapeutic potential of miR-146a-3p in severe asthma and the effect of miR-146a-3p regulated Th17 cells differentiation via MBD2 in vivo. Decreased miR-146a-3p expression was noted in severe asthma patients, in the BECs and in the animal severe asthma models. Moreover, we demonstrated that miR-146a-3p suppressed Th17 cells differentiation by targeting the MBD2. miR-146a-3p overexpression significantly reduced airway hyperresponsiveness, airway inflammation and airway mucus secretion, while also inhibiting Th17 cells response in vivo, which relieved severe asthma. By targeting MBD2 to suppress Th17 cells differentiation, miR-146a-3p provides a potential novel therapeutic for Th17 predominant neutrophilic severe asthma.

Simvastatin Reduces NETosis to Attenuate Severe Asthma by Inhibiting PAD4 Expression.

Objective: Patients with severe asthma respond poorly to corticosteroids, and their care accounts for more than 60% of the total costs attributed to asthma. Neutrophils form neutrophil extracellular traps (NETs), which play a crucial role in severe asthma. Statins have shown anti-inflammatory effects by reducing NETosis. In this study, we investigate if simvastatin can attenuate severe asthma by reducing NETosis and the underlying mechanism. Methods: Mice were concomitantly sensitized with ovalbumin (OVA), house dust mite (HDM), and lipopolysaccharide (LPS) during sensitization to establish a mouse model of severe asthma with neutrophil predominant inflammation (OVA+LPS mice) and treated with or without simvastatin. In inflammatory response, proportions of Th2, Th17, and Treg cells in lung tissue were detected by flow cytometry, and the levels of cytokines, dsDNA, and MPO-DNA in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. Citrullinated histone H3 (CitH3) and peptidyl arginine deiminase 4 (PAD4) in lung tissue were determined by Western blot and immunofluorescence imaging. PAD4 mRNA was determined by quantitative PCR (qPCR). HL-60 cells were differentiated into neutrophil-like cells by 1.25% DMSO. The neutrophil-like cells were treated with or without LPS, and simvastatin was then stimulated with PMA. CitH3 and PAD4 expressions were determined. Results: Sensitization with OVA, HDM, and LPS resulted in neutrophilic inflammation and the formation of NETs in the lungs. Simvastatin treatment reduced the inflammation score, cytokine levels, total cells, and neutrophil counts in the BALF and reduced proportions of Th2 and Th17 but increased Treg cells in lungs of OVA+LPS mice. Simvastatin-treated OVA+LPS mice show reduced NET formation in BALF and lung tissue compared to control mice. Adoptive transfer of neutrophils was sufficient to restore NETosis and neutrophilic inflammation in simvastatin-treated OVA+LPS mice. Simvastatin reduced PAD4 mRNA and protein expression in lung tissues and neutrophils isolated from lungs of OVA+LPS mice and consequent NET formation. In vitro, simvastatin reduced LPS-induced PAD4 upregulation and NETosis in HL-60-differentiated neutrophil-like cells. Furthermore, PAD4-overexpressed lentiviral transduction was sufficient to restore PAD4 protein expression and NETosis in simvastatin-treated HL-60-differentiated neutrophil-like cells. Conclusions: Simvastatin reduces Th17-mediated neutrophilic inflammation and airway hyperreactivity by reducing PAD4 expression and inhibiting NETosis in a mouse model of severe asthma. Severe asthmatic patients with high levels of circulating NETs or sputum NETs may show improved responses to statin treatment.

lncRNA CRNDE Affects Th17/IL-17A and Inhibits Epithelial-Mesenchymal Transition in Lung Epithelial Cells Reducing Asthma Signs.

Background: Asthma treatment is difficult due to disease heterogeneity and comorbidities. In addition, the development of drugs targeting the underlying mechanisms of asthma remains slow. We planned to identify the most upregulated differentially expressed long noncoding RNA in asthma to explore its regulatory patterns and pathways in asthma. Methods: We sensitized mice using a mixture of ovalbumin, house dust mites, and lipopolysaccharide to establish an asthma mouse model. We also sensitized asthma cells with TGF-ß1 in an in vitro model. We performed a microarray analysis to identify the lncRNA with the differential expression level in model mice. We applied hematoxylin and eosin and Masson's trichrome stainings to mouse tissues to quantify the tissue damage extent. Next, we assess the levels of lncRNA CRNDE, miR-29a-3p, TGF-ß1, MCL-1, E-cadherin, vimentin, and snail. We counted the percentages of Th17 cells using flow cytometry. Finally, we performed a dual-luciferase reporter assay to assess the association between lncRNA CRNDE and miR-29a-3p. Results: We successfully established asthma mouse/cell models and selected the lncRNA CRNDE for our study. Transfection of si-CRNDE reduced the degree of injury and inflammation in the mouse model and reversed the TGF-ß1-induced epithelial-mesenchymal transition (EMT) in the cell model. Moreover, the E-cadherin level was upregulated, and the levels of IL-17A, vimentin, snail, and α-SMA were downregulated. We also discovered that lncRNA CRNDE negatively regulated miR-29a-3p and that this one in turn inhibited MCL-1 in mice. After lncRNA CRNDE expression downregulation, the level of miR-29a-3p was increased, and we detected reduced levels of MCL-1 and EMTs. Conclusions: lncRNA CRNDE expression downregulation led to reduced inflammation and reduced lung damage in mice with induced asthma, it inhibited the EMTs of lung epithelial cells via the miR-29a-3p/MCL-1 pathway, and it reduced the levels of Th17/IL-17A cells to reduce asthma signs.

Different clinical characteristics of current smokers and former smokers with asthma: a cross-sectional study of adult asthma patients in China.

Smoking is a trigger for asthma, which has led to an increase in asthma incidence in China. In smokers, asthma management starts with smoking cessation. Data on predictors of smoking cessation in Chinese patients with asthma are scarce. The objective of this study was to find the differences in clinical characteristics between current smokers and former smokers with asthma in order to identify factors associated with smoking cessation. Eligible adults with diagnosed asthma and smoking from the hospital outpatient clinics (n = 2312) were enrolled and underwent a clinical evaluation, asthma control test (ACT), and pulmonary function test. Information on demographic and sociological data, lung function, laboratory tests, ACT and asthma control questionnaire (ACQ) scores was recorded. Patients were divided into a current smokers group and a former smokers group based on whether they had quit smoking. Logistic regression analysis was used to analyze the factors associated with smoking cessation. Of all patients with asthma, 34.6% were smokers and 65.4% were former smokers, and the mean age was 54.5 ± 11.5 years. Compared with current smokers, the former smokers were older, had longer duration of asthma, had higher ICS dose, had more partially controlled and uncontrolled asthma, had more pack-years, had smoked for longer, and had worse asthma control. The logistic regression model showed that smoking cessation was positively correlated with age, female sex, pack-years, years of smoking, partially controlled asthma, uncontrolled asthma, and body mass index (BMI), but was negatively correlated with ACT, FEV1, FEV1%predicted, and widowed status. More than 30% of asthma patients in the study were still smoking. Among those who quit smoking, many quit late, often not realizing they need to quit until they have significant breathing difficulties. The related factors of smoking cessation identified in this study indicate that there are still differences between continuing smokers and former smokers, and these factors should be focused on in asthma smoking cessation interventions to improve the prognosis of patients with asthma.

MBD2 mediates Th17 cell differentiation by regulating MINK1 in Th17-dominant asthma.

Objectives: .Asthma is a highly heterogeneous disease, and T-helper cell type 17 (Th17) cells play a pathogenic role in the development of non-T2 severe asthma. Misshapen like kinase 1 (MINK1) is involved in the regulation of Th17 cell differentiation, but its effect on severe asthma remains unclear. Our previous studies showed that methyl-CpG binding domain protein 2 (MBD2) expression was significantly increased in patients with Th17 severe asthma and could regulate Th17 cell differentiation. The aim of this study was to investigate how MBD2 interacts with MINK1 to regulate Th17 cell differentiation in Th17-dominant asthma. Materials and methods: Female C57BL/6 mice and bronchial epithelial cells (BECs) were used to establish mouse and cell models of Th17-dominant asthma, respectively. Flow cytometry was used to detect Th17 cell differentiation, and the level of IL-17 was detected by enzyme-linked immunosorbent assay (ELISA). Western blot and quantitative real-time PCR (qRT-PCR) were used to detect MBD2 and MINK1 expression. To investigate the role of MBD2 and MINK1 in Th17 cell differentiation in Th17-dominant asthma, the MBD2 and MINK1 genes were silenced or overexpressed by small interfering RNA and plasmid transfection. Results: Mouse and BEC models of Th17-dominant asthma were established successfully. The main manifestations were increased neutrophils in BALF, airway hyperresponsiveness (AHR), activated Th17 cell differentiation, and high IL-17 levels. The expression of MBD2 in lung tissues and BECs from the Th17-dominant asthma group was significantly increased, while the corresponding expression of MINK1 was significantly impaired. Through overexpression or silencing of MBD2 and MINK1 genes, we have concluded that MBD2 and MINK1 regulate Th17 cell differentiation and IL-17 release. Interestingly, MBD2 was also found to negatively regulate the expression of MINK1. Conclusion: Our findings have revealed new roles for MBD2 and MINK1, and provide new insights into epigenetic regulation of Th17-dominant asthma, which is dominated by neutrophils and Th17 cells. This study could lead to new therapeutic targets for patients with Th17-dominant asthma.

Androgen Plays a Potential Novel Hormonal Therapeutic Role in Th17 Cells Predominant Neutrophilic Severe Asthma by Attenuating BECs Regulated Th17 Cells Differentiation via MBD2 Expression.

T helper 17 (Th17) cells subtype of non-T2 asthma is less responsive (resistant) to inhaled corticosteroids (ICS), so also called severe asthma. Methyl-CpG-binding domain protein 2 (MBD2) regulates the differentiation of the Th17 cells, showing the possibility of a therapeutic target in severe asthma. Androgen tends to show beneficial therapeutic effects and is a "hot research topic," but its role in the differentiation and expression of Th17 cells via MBD2 is still unknown. The aim of this study was to evaluate how sex hormone interacts with MBD2 and affects the differentiation and expression of Th17 cells in severe asthma. Here, first, we measured the concentration of androgen, estrogen, and androgen estrogen ratio from subjects and correlated it with severe asthma status. Then, we established an animal model and bronchial epithelial cells (BECs) model of severe asthma to evaluate the role of MBD2 in the differentiation and expression of Th17 cells (IL-17), the therapeutic potential of sex hormones in severe asthma, and the effect of sex hormones in BECs regulated Th17 cells differentiation via MBD2 at the cellular level. Increased MBD2 expression and Th17 cells differentiation were noted in the animal and the BECs severe asthma models. Th17 cell differentiation and expression were MBD2 dependent. Androgen attenuated the differentiation of BECs regulated Th17 cells via MBD2 showing BECs as a therapeutic target of androgen, and these findings postulate the novel role of androgen in Th17 cells predominant neutrophilic severe asthma therapy through targeting MBD2.

Effect of modified Total Body Recumbent Stepper training on exercise capacity and thioredoxin in COPD: a randomized clinical trial.

Exercise intolerance is one of the major symptoms of chronic obstructive pulmonary disease (COPD). Exercise training can benefit COPD patients, but the underlying mechanism remains unclear. The modified Total Body Recumbent Stepper (TBRS, Nustep-T4) can benefit patients with stroke, spinal cord injury and amyotrophic lateral sclerosis. Nevertheless, the effect of TBRS training alone on pulmonary rehabilitation (PR) in COPD patients remains largely unknown. We aimed to explore the effect of TBRS training on exercise capacity and the thioredoxin system (TRXS) in COPD patients to provide a novel rehabilitation modality and new theoretical basis for PR of COPD patients. Ninety stable COPD patients were randomly divided into a control group (NC group) and a TBRS training group (TBRS group), with 45 cases in each group. Subjects in the TBRS training group were scheduled to undergo TBRS endurance training triweekly for 12 weeks under the guidance of a rehabilitation therapist. We assessed the primary outcome: exercise capacity (6-min walking distance, 6MWD); and secondary outcomes: perception of dyspnoea (mMRC, Borg), the COPD assessment test (CAT), the BODE index, pulmonary function, the number of acute exacerbations of COPD and oxidative stress (TRXS) at one-year follow-up. Compared with before the intervention and the control group, after the intervention, the TBRS training group, exhibited an increase in the 6MWD (from 366.92 ± 85.81 to 484.10 ± 71.90, 484.10 ± 71.90 vs 370.63 ± 79.87, P < 0.01), while the scores on the BORG, mMRC, BODE index, CAT, and the number of acute exacerbations of COPD were reduced, and the protein and mRNA expression levels of TRXS was significantly increased (P < 0.01). However, no differences were found in PF parameters in the comparison with before the intervention or between groups. TBRS training can effectively increase exercise capacity, while there are indications that it can alleviate COPD-related dyspnoea and reduce the number of acute exacerbations of COPD. Interestingly, long-term regular TBRS training may reduce oxidative stress associated with COPD to increase exercise capacity.