MAP4K4 exacerbates cardiac microvascular injury in diabetes by facilitating S-nitrosylation modification of Drp1.

Dynamin-related protein 1 (Drp1) is a crucial regulator of mitochondrial dynamics, the overactivation of which can lead to cardiovascular disease. Multiple distinct posttranscriptional modifications of Drp1 have been reported, among which S-nitrosylation was recently introduced. However, the detailed regulatory mechanism of S-nitrosylation of Drp1 (SNO-Drp1) in cardiac microvascular dysfunction in diabetes remains elusive. The present study revealed that mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) was consistently upregulated in diabetic cardiomyopathy (DCM) and promoted SNO-Drp1 in cardiac microvascular endothelial cells (CMECs), which in turn led to mitochondrial dysfunction and cardiac microvascular disorder. Further studies confirmed that MAP4K4 promoted SNO-Drp1 at human C644 (mouse C650) by inhibiting glutathione peroxidase 4 (GPX4) expression, through which MAP4K4 stimulated endothelial ferroptosis in diabetes. In contrast, inhibition of MAP4K4 via DMX-5804 significantly reduced endothelial ferroptosis, alleviated cardiac microvascular dysfunction and improved cardiac dysfunction in db/db mice by reducing SNO-Drp1. In parallel, the C650A mutation in mice abolished SNO-Drp1 and the role of Drp1 in promoting cardiac microvascular disorder and cardiac dysfunction. In conclusion, our findings demonstrate that MAP4K4 plays an important role in endothelial dysfunction in DCM and reveal that SNO-Drp1 and ferroptosis activation may act as downstream targets, representing potential therapeutic targets for DCM.

[Aloperine suppresses TLR4/NF-κB/NLRP3 signaling to ameliorate cigarette smoke-induced injury to human bronchial epithelial cells].

Objective To explore the effects of aloperine (Alo) on cigarette smoke-induced injury in human bronchial epithelial cells and its potential mechanism. Methods After human bronchial epithelial 16HBE cells were co-treated by 100 mL/L cigarette smoke extract (CSE) and various concentrations (50,100 and 200 µmol/L) of Alo, cell viability was assessed using CCK-8 assay. Lactate dehydrogenase (LDH) activity was measured with a related kit. Cell apoptosis was evaluated using the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and Western blot analysis. The levels of inflammatory factors were detected by ELISA. Oxidative stress levels were assessed using 2'7'-dichlorofluorescin diacetate (DCFH-DA) staining. The expression of Toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) signaling-associated proteins was measured by Western blot analysis. After cells were co-treated with 100 mL/L CSE and 200 µmol/L Alo, the aforementioned assays were applied to evaluate the effects of TLR4 overexpression on the TLR4/NF-κB/NLRP3 signaling, LDH activity, apoptosis, inflammatory response and oxidative stress in cells. Results CSE exposure might inhibit 16HBE cell viability, increase LDH activity, apoptosis, inflammatory response and oxidative stress levels and activate TLR4/NF-κB/NLRP3 signaling. Treatment with Alo promoted cell viability, decreased LDH activity, cell apoptosis, inflammation and oxidative stress levels, and inactivated TLR4/NF-κB/NLRP3 signaling. Furthermore, TLR4 overexpression might reverse the protective role of Alo treatment in CSE-induced injury in 16HBE cells. Conclusion Alo may ameliorate CSE-induced injury in human bronchial epithelial cells via inhibiting TLR4/NF-κB/NLRP3 signaling.

L-carnitine alleviates cardiac microvascular dysfunction in diabetic cardiomyopathy by enhancing PINK1-Parkin-dependent mitophagy through the CPT1a-PHB2-PARL pathways.

AIM: To explore the beneficial effects of L-carnitine on cardiac microvascular dysfunction in diabetic cardiomyopathy from the perspectives of mitophagy and mitochondrial integrity. METHODS: Male db/db and db/m mice were randomly assigned to groups and were treated with L-carnitine or a solvent for 24 weeks. Endothelium-specific PARL overexpression was attained via adeno-associated virus serotype 9 (AAV9) transfection. Adenovirus (ADV) vectors overexpressing wild-type CPT1a, mutant CPT1a, or PARL were transfected into endothelial cells exposed to high glucose and free fatty acid (HG/FFA) injury. Cardiac microvascular function, mitophagy, and mitochondrial function were analyzed by immunofluorescence and transmission electron microscopy. Protein expression and interactions were assessed by western blotting and immunoprecipitation. RESULTS: L-carnitine treatment enhanced microvascular perfusion, reinforced endothelial barrier function, repressed the endothelial inflammatory response, and maintained the microvascular structure in db/db mice. Further results demonstrated that PINK1-Parkin-dependent mitophagy was suppressed in endothelial cells suffering from diabetic injury, and these effects were largely alleviated by L-carnitine through the inhibition of PARL detachment from PHB2. Moreover, CPT1a modulated the PHB2-PARL interaction by directly binding to PHB2. The increase in CPT1a activity induced by L-carnitine or amino acid mutation (M593S) enhanced the PHB2-PARL interaction, thereby improving mitophagy and mitochondrial function. In contrast, PARL overexpression inhibited mitophagy and abolished all the beneficial effects of L-carnitine on mitochondrial integrity and cardiac microvascular function. CONCLUSION: L-carnitine treatment enhanced PINK1-Parkin-dependent mitophagy by maintaining the PHB2-PARL interaction via CPT1a, thereby reversing mitochondrial dysfunction and cardiac microvascular injury in diabetic cardiomyopathy.

Exosomal MFI2-AS1 sponge miR-107 promotes non-small cell lung cancer progression through NFAT5.

BACKGROUND: Non-small cell lung cancer is a heterogeneous disease driven by extensive molecular alterations. Exosomes are small vesicles with diameters ranging from 30 to 150 nm released by various cell types and are important mediators of information transmission in tumor cells. Exosomes contain proteins, lipids, and various types of nucleic acids, including miRNAs and even DNA and RNA. MFI2 Antisense RNA 1 (MFI2-AS1) is a long noncoding RNA known to promote cell proliferation, metastasis and invasion in a variety of malignancies. METHODS: The relative expression of MFI2-AS1 in NSCLC tissues was examined using RNA fluorescence in situ hybridization (FISH) staining. Transwell migration and wound healing assays were used to analyze cell migration and invasion abilities. Tube formation is used to assess angiogenic capacity. CCK8 was used to assess cell proliferation ability. RNA immunoprecipitation (RIP) experiments confirmed that MFI2-AS1 acts as a competing endogenous RNA (ceRNA) for miR-107. Dual-luciferase reporter assays were used to identify potential binding between MFI2-miRNA and target mRNA. In vivo experiments were performed by injecting exosomes into subcutaneous tumors to establish animal models. RESULT: Exosomal MFI2-AS1 increases NFAT5 expression by sponging miR-107, which in turn activates the PI3K/AKT pathway. We found that the MFI2-AS1/miR-107/NFAT5 axis plays an important role in exosome-mediated NSCLC progression, is involved in pre-metastatic niche formation, and can be used as a blood-based biomarker for NSCLC metastasis. CONCLUSION: We demonstrate that MFI2-AS1 is upregulated in exosomes secreted by metastatic NSCLC cells and can be transferred to HUVECs, promoting angiogenesis and migration.

Metagenomic next-generation sequencing of radial ultrasound bronchoscopy-guided "cocktail" specimens as an efficient method for the diagnosis of focal pulmonary infections: a randomised study.

BACKGROUND: To investigate the value of metagenomic next-generation sequencing (mNGS) in the diagnosis of focal pulmonary infections by using radial ultrasound bronchoscopic "cocktail" specimens. METHODS: From March 2019 to May 2020, 90 patients with focal pulmonary infections [locatable by a radial probe endobronchial ultrasound (RP-EBUS)] treated by the Department of Respiratory and Critical Care Medicine at the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital (Suzhou, China) were randomly allocated to the "cocktail" group, transbronchial brushing (TBBr) group or bronchoalveolar lavage fluid (BALF) group; 30 patients were assigned to each group. Using the extracted material, the diagnostic efficacy of the mNGS test for pathogen detection was compared across the three groups, and the effect of an antibiotic treatment on detection rate was assessed. RESULTS: The sensitivity of mNGS analysis in cocktail group, TBBr group and BALF group was 90% (27/30), 66.7 (20/30) and 50% (15/30), respectively. The analysis of the sensitivity of the positive test, the detection rate of multiple pathogens, and the effect of antibiotics on the detection rate by mMGS indicated that the "cocktail" group was significantly higher number of samples positive for pathogens than the TBBr group (P<0.05), and the TBBr group was significantly higher number of samples positive for pathogens than the BALF group (P<0.05). CONCLUSIONS: "Cocktail" specimens had high sensitivity in the identification of focal pathogens, and the use of antibiotics had little effect on the results of the mNGS analysis. These clinically valuable results can be used in the diagnosis and treatment of patients with focal pulmonary infections.

Epidemiology and risk factors of chronic obstructive pulmonary disease in Suzhou: a population-based cross-sectional study.

BACKGROUND: The prevalence of chronic obstructive pulmonary disease (COPD) in Suzhou remains still unknown. The aim of this study was to quantify the disease burden and assess the risk factors of COPD. METHODS: This was a population-based, cross-sectional study of adults aged 40 years and older in Suzhou. A total of 4,864 adults were identified from June 2018 to December 2018 and 4,725 adults were finally recruited. Subjects underwent post-bronchodilator spirometry and were diagnosed according to the 2018 Global Initiative for Chronic Obstructive Lung Disease (GOLD). RESULTS: The data from 4,725 adults were ultimately included in the final analysis. The overall prevalence of COPD in subjects aged 40 and older was 12.4%, while it was 12.3% in men and 12.5% in women. Risk factors identified by multivariable logistic analysis were age (P<0.05, OR =2.29, 95% CI, 1.83-2.88) and underweight (BMI <18.5 kg/m2) (P<0.05, OR =1.57, 95% CI, 1.01-2.44). COPD patients also displayed weaker grip strength (P<0.001). Approximately half (50.7%) the COPD patients were asymptomatic, and compared with asymptomatic COPD patients, symptomatic COPD patients were older (69.5 vs. 67.2, P<0.05), smoked more frequently (12.1 vs. 7.1 pack year, P<0.05), had a more severe GOLD stage (stage I 27.0% vs. 39.4%, stage II 50.2% vs. 46.8%, stage III 17.0% vs. 11.1%, stage IV 5.8% vs. 2.7%, P<0.05), and a worse lung function index (FEV1, FVC, PEF, FEF25, FEF50, FEF75, FEF2575) (P<0.05). CONCLUSIONS: COPD was found to be highly prevalent in adults aged 40 years and older in Suzhou. Age and underweight were major risk factors of COPD. Half of the COPD patients were asymptomatic, and displayed decreased lung function upon the onset of respiratory symptoms. Therefore, spirometry screening is essential for the early detection and management of COPD.

NLRP3 inflammasome inhibition attenuates silica-induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells.

Silicosis is an incurable and progressive lung disease characterized by chronic inflammation and fibroblasts accumulation. Studies have indicated a vital role for epithelial-mesenchymal transition (EMT) in fibroblasts accumulation. NLRP3 inflammasome is a critical mediator of inflammation in response to a wide range of stimuli (including silica particles), and plays an important role in many respiratory diseases. However, whether NLRP3 inflammasome regulates silica-induced EMT remains unknown. Our results showed that silica induced EMT in human bronchial epithelial cells (16HBE cells) in a dose- and time-dependent manner. Meanwhile, silica persistently activated NLRP3 inflammasome as indicated by continuously elevated extracellular levels of interleukin-1ß (IL-1ß) and IL-18. NLRP3 inflammasome inhibition by short hairpin RNA (shRNA)-mediated knockdown of NLRP3, selective inhibitor MCC950, and caspase-1 inhibitor Z-YVAD-FMK attenuated silica-induced EMT. Western blot analysis indicated that TAK1-MAPK-Snail/NF-κB pathway involved NLRP3 inflammasome-mediated EMT. Moreover, pirfenidone, a commercially and clinically available drug approved for treating idiopathic pulmonary fibrosis (IPF), effectively suppressed silica-induced EMT of 16HBE cells in line with NLRP3 inflammasome inhibition. Collectively, our results indicate that NLRP3 inflammasome is a promising target for blocking or retarding EMT-mediated fibrosis in pulmonary silicosis. On basis of this mechanism, pirfenidone might be a potential drug for the treatment of silicosis.

Iptakalim influences the proliferation and apoptosis of human pulmonary artery smooth muscle cells.

The aim of the present study was to determine the effect of an ATP-sensitive K+ (KATP) channel opener iptakalim (IPT) on the proliferation and apoptosis of human pulmonary artery smooth muscle cells (HPASMCs), and examine the potential value of IPT to hypoxic pulmonary hyper-tension (HPH) at a cellular level. HPASMCs were divided into the control, ET-1, ET-1+IPT and ET-1+IPT+glibenclamide (GLI) groups. GLI was administered 30 min prior to ET-1 and IPT. The 4 groups were incubated with corresponding reagents for 24 h. Cell viability was evaluated using a CCK-8 assay, cell proliferation by 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and cell apoptosis via the expression of apoptosis-related proteins, i.e., Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) using western blotting. We incubated HPASMCs with varying concentrations of ET-1 for 24, 48 and 72 h, and found that cell survival rate was increased in a dose-dependent manner (P<0.05) rather than in a time-dependent manner (P>0.05). After co-incubation of HPASMCs with varying concentrations of IPT and ET-1 for 24 h, the cell survival rate was decreased in a dose-dependent manner. The cell survival rate in the IPT+ET-1 group was significantly lower than that in the ET-1 group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1 group were higher than those in the control group, and the expression of Bax/Bcl-2 was lower than the control group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1+IPT group were lower than those in the ET-1 group, and the expression of Bax/Bcl-2 was higher than that in the ET-1 group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1+IPT+GLI group were higher than those in the ET-1+IPT group, and the expression of Bax/Bcl-2 was lower than that in the ET-1+IPT group (P<0.05). In conclusion, IPT inhibited ET-1­induced HPASMC proliferation and promoted cell apoptosis. Thus, it may play an important role in the treatment of HPH.

Pulmonary Fungal Diseases in Immunocompetent Hosts: A Single-Center Retrospective Analysis of 35 Subjects.

BACKGROUND: Pulmonary fungal disease is an emerging issue in immunocompetent patients, for whom the characteristics are only partially understood. METHODS: We conducted a single-center retrospective study of histologically verified pulmonary fungal disease in Eastern China from 2006 to 2014 to understand the demographics, clinical manifestations, therapeutic approaches, and factors associated with prognosis in this population. All cases were diagnosed according to the 2008 European Organization for the Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infection Diseases Mycoses Study Group definition criteria. RESULTS: A total of 112 cases of pulmonary fungal diseases were enrolled (35 proven, 16 probable, 61 possible), and we analyzed the 35 patients with histologically proven pulmonary fungal diseases in this study. The main fungal species identified were Aspergillus (51.4 %), Cryptococcus (22.9 %), and Mucor (2.4 %). Treatment consisted of antifungal therapeutic agents (54.3 %), surgery and postsurgical agents (25.7 %), or surgery alone (14.3 %). The overall crude mortality rate was 14.3 %, and the mortality due to pulmonary fungal infections was 2.9 %. Significant predictors of mortality by univariate analysis were hypoalbuminemia (P = 0.005), cancer (P = 0.008), and positive culture (P = 0.044). Additionally, hypoalbuminemia was the only risk factor for mortality by multivariate analysis (RR = 7.56, 95 % CI 1.38-41.46). CONCLUSION: Pulmonary fungal disease in immunocompetent patients, with Aspergillus as the most common identified species, had a prognosis that was influenced by the level of serum albumin.

Activation of NLRP3 inflammasome enhances the proliferation and migration of A549 lung cancer cells.

Lung cancer is the leading cause of cancer death, and it is widely accepted that chronic inflammation is an important risk for the development of lung cancer. Now, it is recognized that the nucleotide-binding and oligomerization domain (NOD) like receptors (NLRs)-containing inflammasomes are involved in cancer-related inflammation. This study was designed to investigate the effects of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome on the proliferation and migration of lung adenocarcinoma cell line A549. Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, scratch assay, and Transwell migration assay, we showed that activation of the NLRP3 inflammasome by LPS+ATP enhanced the proliferation and migration of A549 cells. Western blot analysis showed that activation of phosphorylation of Akt, ERK1/2, CREB and the expression of Snail increased, while the expression of E-cadherin decreased after the activation of NLRP3 inflammasome. Moreover, these effects were inhibited by the following treatments: i) downregulating the expression of NLRP3 by short hairpin RNA (shRNA) interference, ii) inhibiting the activation of NLRP3 inflammasome with a caspase-1 inhibitor, iii) blocking the interleukin-1ß (IL-1ß) and IL-18 signal transduction with IL-1 receptor antagonist (IL-1Ra) and IL-18 binding protein (IL-18BP). Collectively, these results indicate that NLRP3 inflammasome plays a vital role in regulating the proliferation and migration of A549 cells and it might be a potential target for the treatment of lung cancer.

Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration.

Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (KATP) channels have been identified in ASMCs. Mount evidence has suggested that KATP channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K(+) channels triggers K(+) efflux, which leading to membrane hyperpolarization, preventing Ca(2+)entry through closing voltage-operated Ca(2+) channels. Intracellular Ca(2+) is the most important regulator of muscle contraction, cell proliferation and migration. K(+) efflux decreases Ca(2+) influx, which consequently influences ASMCs proliferation and migration. As a KATP channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca(2+)/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective KATP channel antagonist. These findings provide a strong evidence to support that Ipt antagonize the proliferating and migrating effects of PDGF-BB on human ASMCs through opening KATP channels. Altogether, our results highlighted a novel profile of Ipt as a potent option against the airway remodeling in chronic airway diseases.

One-pot catalytic conversion of cellulose into polyols with Pt/CNTs catalysts.

A series of Pt nanoparticles supported on carbon nanotubes (CNTs) were synthesized using the incipient-wetness impregnation method. These catalysts were characterized by X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Raman spectroscopy, and transmission electron microscope (TEM) techniques. The characterization results indicate that the Pt nanoparticles were highly dispersed on the surface of the CNTs, and the mean size was less than 5 nm. These catalysts were utilized to convert cellulose to hexitol, ethylene glycerol (EG), and 1,2-propylene glycol (1,2-PG) under low H2 pressure. The total yields were as high as 71.4% for EG and 1,2-PG using 1Pt/CNTs as the catalyst in the hydrolytic hydrogenation of cellulose under mild reaction conditions.

[Analysis of pathogenic spectrum and risk factors in 165 non-transplant patients with invasive fungal disease].

OBJECTIVE: To describe the clinical characteristics of and risk factors for invasive fungal disease, and therefore to improve the early diagnosis and treatment of fungal infections. METHODS: The clinical data of invasive fungal disease in 165 patients without transplantation from 2006 to 2012 in the First Affiliated Hospital of Nanjing Medical University were retrospectively analyzed. The diagnosis was based on the following guidelines: diagnosis and treatment guidelines of critically ill patients with invasive fungal infection (2007), diagnostic criteria and treatment principle of invasive fungal infection in patients with hematopathy/malignant tumors (fourth edition, 2013), diagnostic criteria and treatment principle of invasive pulmonary fungal infection (draft, 2006). RESULTS: Invasive fungal disease was mostly diagnosed in the respiratory department (31.5%). The major pathogens were cryptococcus (48.3%), aspergillus (31.7%) and followed by mucor (5.9%). The most common symptoms included cough, haemoptysis, and fever. Radiological Findings were non-specific, nodules or opacities being more common as compared to classical aspergilloma, halo sign, and crescent sign. The most common underlying diseases were diabetes (15.8%), chronic obstructive pulmonary disease (13.3%), and malignant hematological disease (10.3%). Moreover, 66.1% cases of invasive fungal disease were accompanied by one or more risk factors (eg. administration of antibiotics more than 7 days, invasive operations, and therapy with long-term glucocorticoids or immunosuppressant drugs). The mortality of invasive fungal disease with more than 2 risk factors was 10.6%. CONCLUSIONS: The most common pathogens of invasive fungal disease in non-transplant patients were cryptococcus, aspergillus and mucor. The lung and the brain were the mostly involved organs. Compared to cryptococcus, invasive fungal disease caused by other fungal pathogens mainly occurred in patients with serious underlying diseases and risk factors.