Hydrogen sulfide inhibits alveolar type II cell senescence and limits pulmonary fibrosis via promoting MDM2-mediated p53 degradation.

AIM: Senescence of alveolar type II (AT2) cells is an important driver of pulmonary fibrosis. This study aimed to investigate whether and how dysregulation of hydrogen sulfide (H2 S) production affected AT2 cell senescence, and then explored the effect of H2 S on the communication between AT2 and fibroblasts. METHODS: ICR mice were intratracheally administered with bleomycin (3 mg/kg). Sodium hydrosulfide (NaHS, 28 µmol/kg/d) was intraperitoneally injected for 2 weeks. The H2 S-generating enzyme cystathionine-ß-synthase (CBS) knockout heterozygous (CBS+/- ) mice were used as a low H2 S production model. RESULTS: Analysis of microarray datasets revealed downregulation of H2 S-generating enzymes in lung tissues of patients with pulmonary fibrosis. Decreased H2 S production was correlated with higher levels of cell senescence markers p53 and p21 in bleomycin-induced lung fibrosis. CBS+/- mice exhibited increased levels of p53 and p21. The numbers of AT2 cells positive for p53 and p21 were increased in CBS+/- mice as compared to control mice. H2 S donor NaHS attenuated bleomycin-induced AT2 cell senescence both in vivo and in vitro. H2 S donor suppressed bleomycin-induced senescence-associated secretory phenotype (SASP) of AT2 cells via inhibiting p53/p21 pathway, consequently suppressing proliferation and myofibroblast transdifferentiation of fibroblasts. Mechanically, H2 S suppressed p53 expression by enhancing the mouse double-minute 2 homologue (MDM2)-mediated ubiquitination and degradation of p53. CONCLUSION: H2 S inactivated p53-p21 pathway, consequently suppressing AT2 cell senescence as well as cell communication between senescent AT2 cells and fibroblasts. Aberrant H2 S synthesis may contribute to the development of pulmonary fibrosis through promoting the activation loop involving senescent AT2 cells and activated fibroblasts.

Upregulation of KLK8 contributes to CUMS-induced hippocampal neuronal apoptosis by cleaving NCAM1.

Neuronal apoptosis has been well-recognized as a critical mediator in the pathogenesis of depressive disorders. Tissue kallikrein-related peptidase 8 (KLK8), a trypsin-like serine protease, has been implicated in the pathogenesis of several psychiatric disorders. The present study aimed to explore the potential function of KLK8 in hippocampal neuronal cell apoptosis associated with depressive disorders in rodent models of chronic unpredictable mild stress (CUMS)-induced depression. It was found that depression-like behavior in CUMS-induced mice was associated with hippocampal KLK8 upregulation. Transgenic overexpression of KLK8 exacerbated, whereas KLK8 deficiency attenuated CUMS-induced depression-like behaviors and hippocampal neuronal apoptosis. In HT22 murine hippocampal neuronal cells and primary hippocampal neurons, adenovirus-mediated overexpression of KLK8 (Ad-KLK8) was sufficient to induce neuron apoptosis. Mechanistically, it was identified that the neural cell adhesion molecule 1 (NCAM1) may associate with KLK8 in hippocampal neurons as KLK8 proteolytically cleaved the NCAM1 extracellular domain. Immunofluorescent staining exhibited decreased NCAM1 in hippocampal sections obtained from mice or rats exposed to CUMS. Transgenic overexpression of KLK8 exacerbated, whereas KLK8 deficiency largely prevented CUMS-induced loss of NCAM1 in the hippocampus. Both adenovirus-mediated overexpression of NCAM1 and NCAM1 mimetic peptide rescued KLK8-overexpressed neuron cells from apoptosis. Collectively, this study identified a new pro-apoptotic mechanism in the hippocampus during the pathogenesis of CUMS-induced depression via the upregulation of KLK8, and raised the possibility of KLK8 as a potential therapeutic target for depression.

Protein Expression Profiles in Exosomes of Bovine Mammary Epithelial Cell Line MAC-T Infected with Staphylococcus aureus.

Mastitis is a common and widespread infectious disease in dairy farms around the world, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis in dairy cows. S. aureus can activate inflammatory signaling pathways in bovine mammary epithelial cells. Exosomes produced by cells can directly transfer pathogen-related molecules from cell to cell, thus affecting the process of infection. Protein is the material basis of the immune defense function in the body; therefore, a comprehensive comparison of proteins in exosomes derived from S. aureus-infected (SA group) and normal (control group [C group]) bovine mammary epithelial MAC-T cells was performed using shotgun proteomics by a DIA approach. A total of 7,070 proteins were identified and quantified. Compared with the C group, there were 802 differentially expressed proteins (DEPs) identified in the SA group (absolute log2 fold change [|log2FC|] of ≥0.58; false discovery rate [FDR] of <0.05), among which 325 proteins were upregulated and 477 were downregulated. The upregulated proteins, including complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK), are involved mostly in host defense against pathogens, inflammation, and cell structure maintenance. KEGG enrichment analysis indicated that DEPs in S. aureus infection were involved in the complement and coagulation cascade, phagosome, extracellular matrix (ECM)-receptor interaction, and focal adhesion pathways. The results of this study provide novel information about proteins in the exosomes of MAC-T cells infected with S. aureus and could contribute to an understanding of the infectious mechanism of bovine mastitis. IMPORTANCE Mastitis is a widespread infectious disease in dairy farms, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis. Exosomes contain proteins, lipids, and nucleic acids, which are involved in many physiological and pathological functions. The expression of proteins in exosomes derived from bovine mammary epithelial cells infected by S. aureus is still barely understood. These results provide novel information about MAC-T-derived exosomal proteins, reveal insights into their functions, and lay a foundation for further studying the biological function of exosomes during the inflammatory response.

Matrine Attenuates Lung Injury by Modulating Macrophage Polarization and Suppressing Apoptosis.

INTRODUCTION: Persistent lung inflammation is a characteristic of sepsis-induced lung injury. Matrine, the active ingredient from Sophora flavescens, has exhibited anti-inflammatory activities. This study investigated the effects of prophylactic administration of matrine on macrophage polarization, apoptosis, and tissue injury in a cecal ligation and puncture (CLP)-induced murine lung injury model. METHODS: Mice were randomly allocated into four groups: Sham, CLP, Sham + Matrine, and CLP + Matrine. Lung tissues were collected at 24 h post-CLP. Histopathology and immunofluorescence analysis were performed to evaluate lung injury and macrophage infiltration in the lung, respectively. Caspase-3 activities, TUNEL staining, and anti-apoptotic proteins were examined to assess apoptosis. To determine the mechanism of action of matrine, protein levels of Sirtuin 1 (SIRT1), nuclear factor κB (NF-κB), p53 and the messenger RNA levels of p53-mediated proapoptotic genes were examined to elucidate the associated signaling pathways. RESULTS: Histopathological evaluation showed that matrine prophylaxis attenuated sepsis-induced lung injury. Matrine prophylaxis attenuated sepsis-induced infiltration of the total population of macrophages in the lung. Matrine inhibited M1 macrophage infiltration, but increased M2 macrophage infiltration, thus resulting in a decrease in the proportion of M1 to M2 macrophages in septic lung. Sepsis-induced lung injury was associated with apoptotic cell death as evidenced by increases in caspase-3 activity, TUNEL-positive cells, and decreases in antiapoptotic proteins, all of which were reversed by matrine prophylaxis. Matrine restored sepsis-induced downregulation of SIRT1 and deacetylation of NF-κB p65 subunit and p53, thus inactivating NF-κB pathway and suppressing p53-induced proapoptotic pathway in septic lung. CONCLUSIONS: In summary, this study demonstrated that matrine exhibited pro-M2 macrophage polarization and antiapoptotic effects in sepsis-induced lung injury, which might be, at least partly, due to the modulation of SIRT1/NF-κB and SIRT1/p53 pathways.

Cancer-derived exosomal miR-197-3p confers angiogenesis via targeting TIMP2/3 in lung adenocarcinoma metastasis.

Cancer-derived exosomal miRNAs are implicated in tumorigenesis and development of lung adenocarcinoma (LUAD). The objective of this study is to unravel the biological function of exosomal miR-197-3p in LUAD metastasis. qRT-PCR showed that elevated miR-197-3p in LUAD tissues was positively correlated with LUAD metastasis. CCK-8, tube formation, transwell and wound healing assays revealed that exosomal miR-197-3p from LUAD cells promoted the proliferation, angiogenesis and migration of HUVECs in vitro. LUAD cells-derived exosomal miR-197-3p also facilitated tumor growth and angiogenesis in LUAD cells-derived tumor xenograft model. TIMP2 and TIMP3 were identified as target genes of miR-197-3p in HUVECs by bioinformatics analysis and luciferase reporter assay. Functional studies illustrated that exosomal miR-197-3p promoted angiogenesis and migration via targeting TIMP2 and TIMP3 in HUVECs. In vivo data further supported that exosomal miR-197-3p promoted lung metastasis via TIMP2/3-mediated angiogenesis. In conclusion, LUAD cells-derived exosomal miR-197-3p conferred angiogenesis via targeting TIMP2/3 in LUAD metastasis.

Keep a watchful eye on methionine adenosyltransferases, novel therapeutic opportunities for hepatobiliary and pancreatic tumours.

Methionine adenosyltransferases (MATs) synthesize S-adenosylmethionine (SAM) from methionine, which provides methyl groups for DNA, RNA, protein, and lipid methylation. MATs play a critical role in cellular processes, including growth, proliferation, and differentiation, and have been implicated in tumour development and progression. The expression of MATs is altered in hepatobiliary and pancreatic (HBP) cancers, which serves as a rare biomarker for early diagnosis and prognosis prediction of HBP cancers. Independent of SAM depletion in cells, MATs are often dysregulated at the transcriptional, post-transcriptional, and post-translational levels. Dysregulation of MATs is involved in carcinogenesis, chemotherapy resistance, T cell exhaustion, activation of tumour-associated macrophages, cancer stemness, and activation of tumourigenic pathways. Targeting MATs both directly and indirectly is a potential therapeutic strategy. This review summarizes the dysregulations of MATs, their proposed mechanism, diagnostic and prognostic roles, and potential therapeutic effects in context of HBP cancers.

[Establishment of a three-dimensional organoid culture system for mouse type 2 alveolar epithelial cells].

The purpose of this study was to establish a three-dimensional (3D) organoid culture system for type 2 alveolar epithelial (AT2) cells in mice. AT2 cells were isolated from ICR mouse lung and purified by enzymatic digestion and MicroBeads sorting. The purity of AT2 cells was determined by immunofluorescence (IF) staining using an antibody against proSPC. The AT2 differentiation was examined by IF staining with proSPC/HopX and proSPC/T1α antibodies, and proliferation of AT2 cells was assessed by EdU incorporation assays after two-dimensional (2D) culture for 8 days. In addition, AT2 cells were co-cultured with mouse lung fibroblasts (Mlg) in three-dimensional (3D) culture system. After 13 days of co-culture, the organoids were fixed in 2% paraformaldehyde for histological analysis and IF staining. The results showed that the purity of the AT2 cells was over 95%, as assessed by proSPC staining. 2D cultured AT2 cells were negative for EdU staining, which indicates that no proliferation occurs. proSPC expression was gradually disappeared, whereas T1α and HopX expression was gradually increased after 3, 5 and 8 days of culture. In 3D culture system, the alveolar organoids were formed after co-culturing AT2 cells with Mlg for 4 days. Histological analysis showed that alveolar organoids displayed a hollow morphology. proSPC was highly expressed in the peripheral cells, whereas type 1 alveolar epithelial (AT1) cells transdifferentiated from AT2 cells expressing HopX were mainly located in the interior of organoid bodies after 13 days. Some of the proSPC-positive AT2 cells located in the outer circle of alveolar organoids were stained positive for both proSPC and EdU, indicating that the AT2 cells in the alveolar organoids were proliferative. These results showed that the 3D organoid culture system of mouse AT2 cells was successfully established.

Concurrent alcoholic cirrhosis and malignant peritoneal mesothelioma in a patient: A case report.

BACKGROUND: Malignant peritoneal mesothelioma (MPM) originates from the mesothelial and subcutaneous cells of the abdominal cavity. Its diagnose is difficult due to its nonspecific and vague symptoms, and it should be differentiated from alcoholic cirrhosis and liver and pancreatic cancers. Misdiagnosis and missed diagnosis can easily occur when MPM presents with other diseases. To the best of our knowledge, no case of MPM concurrent with alcoholic cirrhosis has been reported. CASE SUMMARY: A 63-year-old man presented to our hospital with abdominal distension for 20days. He had a history of alcohol consumption for nearly 30 years and no history of special drug use or toxic exposure. After treatment for alcoholic cirrhosis in a community hospital, his symptoms did not improve significantly. The patient underwent exploratory laparotomy and surgical resection. Pathologic examination showed an epithelioid MPM. He was treated with chemotherapy and intraperitoneal hyperthermic perfusion after surgery. Currently, he is in a stable condition and tumor recurrence has not occurred. CONCLUSION: Misdiagnosis and missed diagnosis of MPM can easily occur because of its insidious onset. Therefore, there is a need to understand. MPM in clinical practice, make the correct diagnosis, and provide timely and effective treatment.

Integrating network pharmacology and experimental models to investigate the efficacy of QYHJ on pancreatic cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The Qingyihuaji decoction (QYHJ) is composed of seven herbs: Scutellaria barbata D.Don (Banzhilian, HSB), Gynostemma pentaphyllum (Thunb.) Makino (Jiaogulan, GP), Oldenlandia diffusa (Willd.) Roxb. (Baihuasheshecao, HDH), Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi, GL), Myristica fragrans Houtt. (Doukou, AK), and Amorphophallus kiusianus (Makino) Makino (Sheliugu, RA), and Coix lacryma-jobi var. ma-yuen (Rom.Caill.) Stapf (Yiyiren, CL). QYHJ has been reported to exhibit clinical efficacy in the treatment of pancreatic adenocarcinoma (PAAD). However, the molecular mechanism remains unclear. AIM OF THE STUDY: This study explores the therapeutic mechanism of QYHJ in the treatment of PAAD using network pharmacology to identify related targets and pathways in vivo and in vitro. MATERIALS AND METHODS: The bioactive compounds of QYHJ were retrieved and screened using the ADME network pharmacology approach, followed by compound-target prediction and overlapping genes between PAAD oncogenes and QYHJ target genes. The compound-target-pathway network was established using The KEGG pathway, GO analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis to identify potential action pathways. The effects of QYHJ on PAAD were evaluated in vivo and in vitro, and the predicted targets and potential pathways related to QYHJ in PAAD treatment were evaluated using qRT-PCR and immunoblotting. RESULTS: A total of 68 bioactive compounds of QYHJ fulfilled the ADME screening criterion, and their respective 242 target genes were retrieved. The compound-target-disease network identified 11 possible target genes. The KEGG pathway analysis showed significant enrichment of pathways in cancers, involving regulating cancer-related pathways of inflammation, oxidative stress, and apoptosis. Furthermore, QYHJ inhibited PAAD growth in vivo; suppressed cell proliferation, invasion, and migration of PAAD; and induced cellular apoptosis in vitro. The qRT-PCR results showed that QYHJ suppressed the mRNA expression of ICAM1, VCAM1, and Bcl2, and increased that of HMOX1 and NQO1. Immunoblotting revealed changes in the PI3K/AKT/mTOR, Keap1/Nrf2/HO-1/NQO1, and Bcl2/Bax pathways upon QYHJ treatment. CONCLUSIONS: QYHJ can suppress PAAD growth and progression through various mechanisms, including anti-inflammation and apoptosis-induction.

Upregulation of FOXO1 contributes to lipopolysaccharide-induced pulmonary endothelial injury by induction of autophagy.

Background: Autophagy is activated during the pathogenesis of endothelial dysfunction and sepsis-associated acute lung injury (ALI). This study aimed to investigate whether autophagy affected endothelial barrier dysfunction and lung injury in a murine model of lipopolysaccharide (LPS)-induced ALI, and then further clarify whether forkhead box O1 (FOXO1), an autophagy-related transcriptional factor, contributed to autophagy activation and ALI induced by LPS. Methods: Male C57BL/6 mice were treated with LPS (30 mg/kg), and then were allocated to a control group and an LPS group with or without FOXO1 inhibitor (AS1842856) treatment, respectively. Primary cultured mouse lung vascular endothelial cells (MLVECs) were treated with LPS, autophagy inhibitor 3-methyladenine (3-MA), AS1842856, and small interfering RNA (siRNA) targeting autophagy-related gene 5 (ATG5) or FOXO1. Endothelial autophagic flux was assessed by transfection of MLVECs with red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3) adenovirus. Endothelial permeability was analyzed by the diffusion of fluorescein isothiocyanate-carboxymethyl (FITC)-dextran through the endothelial monolayer. Evans blue albumin tracer was used to measure the pulmonary transvascular permeability, and hematoxylin and eosin (H&E) staining was used to observe pathological changes in the lung tissues. Immunofluorescence staining was also used to detect the expression of zonula occludens-1 (ZO-1) and FOXO1. Results: This study found autophagy induction in lung tissues of endotoxemic mice and LPS-treated MLVECs, as evidenced by elevated expression of light chain 3 II (LC3-II) and Unc-51-like kinase (ULK1) and autophagic flux. LPS treatment decreased vascular endothelial (VE)-cadherin and ZO-1 expression and increased endothelial permeability in MLVECs, which were significantly alleviated by autophagy inhibitor 3-MA and ATG5 siRNA. It was found that both phosphorylated FOXO1 and FOXO1 were upregulated in the lung tissues of endotoxemic mice and LPS-treated MLVECs. Both FOXO1 inhibitor AS1842856 and FOXO1 siRNA suppressed LPS-induced autophagy and endothelial cell injury in MLVECs. Moreover, FOXO1 inhibition profoundly alleviated autophagy, lung endothelial hyperpermeability, and ALI in endotoxemic mice. Conclusions: This work demonstrated that FOXO1 upregulation is an important contributor to LPS-induced autophagy in pulmonary VE cells. The detrimental effects of FOXO1 in endotoxemia-associated endothelial dysfunction and ALI are partly due to its potent pro-autophagic property. Inhibition of FOXO1 may be a potential therapeutic option for the treatment of ALI.

[Application value of imaging examination in the diagnosis of Muller-Weiss disease].

OBJECTIVE: To analyze radiological characteristics of Muller-Weiss disease, evaluate the clinical value of the imaging examination in diagnosis of Muller-Weiss disease. METHODS: The imaging data of 26 patients with Muller-Weiss disease were collected from September 2015 to August 2020, including 7 males and 19 females, aged 43 to 68 years old with an average of (52.7±4.6) years old. In the X-ray examination observed the shape and position of the navicular bone. The talar-first metatarsal angle(TFM) was measured on the weight-bearing anteroposterior radiograph. The arch angle and angle between mid-axis of talus and mid-axis of the first metatarsal(Meary angle) were measured on the weight-bearing lateral radiographs. The morphology, density, adjacent joint space and position of the navicular bone were evaluated by computed tomography(CT), and magnetic resonance imaging(MRI) was used to observe the shape, signal, cartilage and surrounding soft tissue changes of the navicular bone. RESULTS: Among 26 patients, 21 cases were unilateral and 5 cases were bilateral;X-ray examination showed that the lateral part of navicular bone of foot was compressed and flattened, showing"comma like"or"drop like", navicular moved to the medial side, partial fragmentation of bone, peripheral articular hyperplasia, uneven density and narrowing of relationship gap. According to Meary angle and deformity degree of the affected foot on the lateral X-ray of the load-bearing foot, Maceira staging was performed. There were 0 cases in stageⅠ, 2 cases in stage Ⅱ, 11 cases in stage Ⅲ, 9 cases in stage Ⅳand 4 cases in stage Ⅴ. CT examination showed bone fragmentation, medial displacement of navicular bone and formation of the talocalcaneal joint. MRI examination showed the irregular shape and uneven signal of navicular bone, narrowing of joint space, talocalcaneal joint surface hyperplasia and cartilage destruction, tarsal joint effusion and swelling of surrounding soft tissue. CONCLUSION: Muller-Weiss disease has specific imaging manifestation, and an accurate diagnosis can be made based on the patient's age, gender, and clinincal history. Preoperative imaging examination can stage the disease, help clinicians to formulate better surgical plans, and postoperative imaging examination can better evaluate the surgical effect.

Coagulants put phosphate-accumulating organisms at a competitive disadvantage with glycogen-accumulating organisms in enhanced biological phosphorus removal system.

Enhanced biological phosphorus removal (EBPR) process is susceptible to the changed operation condition, which results in an unstable treatment performance. In this work, long-term effect of coagulants addition, aluminum salt for the reactor R1 and iron salt for the reactor R2, on EBPR systems was comprehensively evaluated. Results showed that during the initial 30 days' coagulant addition, effluent chemical oxygen demand and phosphorus can be reduced below 25 and 0.5 mg·L-1, respectively. Further supply of metal salts would stimulate microbial extracellular polymeric substance excretion and induce reactive oxygen species accumulation, which destroyed the cell membrane integrity and deteriorated the phosphorus removal performance. Moreover, coagulants would decrease the relative abundance of Candidatus Accumulibacter while increase the relative abundance of Candidatus Competibacter, leading phosphors accumulating organisms in a disadvantage position. The results of this work might be valuable for the operation of chemical assisted biological phosphorus removal bioreactor.

Exercise training ameliorates early diabetic kidney injury by regulating the H2 S/SIRT1/p53 pathway.

Exercise training exerts protective effects against diabetic nephropathy. This study aimed to investigate whether exercise training could attenuate diabetic renal injury via regulating endogenous hydrogen sulfide (H2 S) production. First, C57BL/6 mice were allocated into the control, diabetes, exercise, and diabetes + exercise groups. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). Treadmill exercise continued for four weeks. Second, mice was allocated into the control, diabetes, H2 S and diabetes + H2 S groups. H2 S donor sodium hydrosulfide (NaHS) was intraperitoneally injected once daily for four weeks. STZ-induced diabetic mice exhibited glomerular hypertrophy, tissue fibrosis and increased urine albumin levels, urine protein- and albumin-to-creatinine ratios, which were relieved by exercise training. Diabetic renal injury was associated with apoptotic cell death, as evidenced by the enhanced caspase-3 activity, the increased TdT-mediated dUTP nick-end labeling -positive cells and the reduced expression of anti-apoptotic proteins, all of which were attenuated by exercise training. Exercise training enhanced renal sirtuin 1 (SIRT1) expression in diabetic mice, accompanied by an inhibition of the p53-#ediated pro-apoptotic pathway. Furthermore, exercise training restored the STZ-mediated downregulation of cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) and the reduced renal H2 S production. NaHS treatment restored SIRT1 expression, inhibited the p53-mediated pro-apoptotic pathway and attenuated diabetes-associated apoptosis and renal injury. In high glucose-treated MPC5 podocytes, NaHS treatment inhibited the p53-mediated pro-apoptotic pathway and podocyte apoptosis in a SIRT1-dependent manner. Collectively, exercise training upregulated CBS/CSE expression and enhanced the endogenous H2 S production in renal tissues, thereby contributing to the modulation of the SIRT1/p53 apoptosis pathway and improvement of diabetic nephropathy.

Novel Insights into the Potential Diagnostic Value of Circulating Exosomal IncRNA-Related Networks in Large Artery Atherosclerotic Stroke.

Exosomes show diagnostic and therapeutic promise as carriers of ncRNAs in diseases. LncRNAs in exosomes have been identified as being stable and avoided degradation by nucleolytic enzymes. Although lncRNAs have been confirmed to be important in cancers, no studies for exo-lncRNAs have been reported in LAA stroke. High-throughput sequencing was performed to detect the differential expression profiles of lncRNAs in five paired plasma-derived exosome samples from patients with LAA stroke and controls (matched on vascular risk factors). Exo-lncRNA-associated networks were predicted with a combination of multiple databases. The expression of the selected genes in the networks was confirmed by qRT-PCR in a validation set (LAA vs. controls = 30:30). Furthermore, ROC analysis was used to evaluate the diagnostic performance of the lncRNA-related networks. A total of 1,020 differentially expressed lncRNAs were identified in LAA stroke patients. GO and KEGG pathway analyses indicated that their target genes are involved in atherosclerosis-related pathways, including inflammation, cell adhesion, and cell migration. qRT-PCR confirmed that the expression trend of differential expressed genes was consistent with RNA-seq. Furthermore, the AUCs of the lnc_002015-related network and lnc_001350-related network were 0.959 and 0.97, respectively, in LAA stroke. Our study showed the differential expression of lncRNAs in plasma exosomes and presented related diagnostic networks for LAA stroke for the first time. The results suggested that exosomal lncRNA-related networks could be potential diagnostic tools in LAA stroke.

[Study of salivary metabonomics in patients with oral squamous cell carcinoma and leukoplakia].

PURPOSE: The objective of this study was to detect and analyze the existence of differential metabolites from saliva of patients with oral squamous cell carcinoma(OSCC) by metabonomics method, and to evaluate its application value on clinical diagnosis or screening of OSCC. METHODS: The collected saliva samples for OSCC patients, oral leukoplakiac(OLK) patients who were pathologically diagnosed and met the inclusion criteria were analyzed using metabonomics methods (GC-TOF-MS). The results were analyzed by principal component analysis and orthogonal partial least squares discriminant analysis using SIMCA software. RESULTS: The metabolic profiles of OSCC, OLK and healthy control group showed significant differences (P<0.05). In total, 15 typical differential metabolites among the three groups were detected. Further study focusing on metabolic pathway revealed imbalance of protein, energy and lipid metabolism between OSCC and OLK patients. Abnormal tricarboxylic acid cycle was detected as well. CONCLUSIONS: Metabonomics methods is feasible for differential metabolites analysis,15 differential metabolites were detected in OSCC,OLK patients and healthy people. These findings will contribute to the early screening of oral squamous cell carcinoma.

A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis.

Rationale: Among all the diabetic complications, diabetic cardiomyopathy, which is characterized by myocyte loss and myocardial fibrosis, is the leading cause of mortality and morbidity in diabetic patients. Tissue kallikrein-related peptidases (KLKs) are secreted serine proteases, that have distinct and overlapping roles in the pathogenesis of cardiovascular diseases. However, whether KLKs are involved in the development of diabetic cardiomyopathy remains unknown.The present study aimed to determine the role of a specific KLK in the initiation of endothelial-to-mesenchymal transition (EndMT) during the pathogenesis of diabetic cardiomyopathy. Methods and Results-By screening gene expression profiles of KLKs, it was found that KLK8 was highly induced in the myocardium of mice with streptozotocin-induced diabetes. KLK8 deficiency attenuated diabetic cardiac fibrosis, and rescued the impaired cardiac function in diabetic mice. Small interfering RNA (siRNA)-mediated KLK8 knockdown significantly attenuated high glucose-induced endothelial damage and EndMT in human coronary artery endothelial cells (HCAECs). Diabetes-induced endothelial injury and cardiac EndMT were significantly alleviated in KLK8-deficient mice. In addition, transgenic overexpression of KLK8 led to interstitial and perivascular cardiac fibrosis, endothelial injury and EndMT in the heart. Adenovirus-mediated overexpression of KLK8 (Ad-KLK8) resulted in increases in endothelial cell damage, permeability and transforming growth factor (TGF)-ß1 release in HCAECs. KLK8 overexpression also induced EndMT in HCAECs, which was alleviated by a TGF-ß1-neutralizing antibody. A specificity protein-1 (Sp-1) consensus site was identified in the human KLK8 promoter and was found to mediate the high glucose-induced KLK8 expression. Mechanistically, it was identified that the vascular endothelial (VE)-cadherin/plakoglobin complex may associate with KLK8 in HCAECs. KLK8 cleaved the VE-cadherin extracellular domain, thus promoting plakoglobin nuclear translocation. Plakoglobin was required for KLK8-induced EndMT by cooperating with p53. KLK8 overexpression led to plakoglobin-dependent association of p53 with hypoxia inducible factor (HIF)-1α, which further enhanced the transactivation effect of HIF-1α on the TGF-ß1 promoter. KLK8 also induced the binding of p53 with Smad3, subsequently promoting pro-EndMT reprogramming via the TGF-ß1/Smad signaling pathway in HCAECs. The in vitro and in vivo findings further demonstrated that high glucose may promote plakoglobin-dependent cooperation of p53 with HIF-1α and Smad3, subsequently increasing the expression of TGF-ß1 and the pro-EndMT target genes of the TGF-ß1/Smad signaling pathway in a KLK8-dependent manner. Conclusions: The present findings uncovered a novel pro-EndMT mechanism during the pathogenesis of diabetic cardiac fibrosis via the upregulation of KLK8, and may contribute to the development of future KLK8-based therapeutic strategies for diabetic cardiomyopathy.

DRD1 downregulation contributes to mechanical stretch-induced lung endothelial barrier dysfunction.

Rationale: The lung-protective effects of dopamine and its role in the pathology of ventilator-induced lung injury (VILI) are emerging. However, the underlying mechanisms are still largely unknown. Objective: To investigate the contribution of dopamine receptor dysregulation in the pathogenesis of VILI and therapeutic potential of dopamine D1 receptor (DRD1) agonist in VILI. Methods: The role of dopamine receptors in mechanical stretch-induced endothelial barrier dysfunction and lung injury was studied in DRD1 knockout mice, in isolated mouse lung vascular endothelial cells (MLVECs), and in lung samples from patients who underwent pulmonary lobectomy with mechanical ventilation for different time periods. Measurements and Main Results: DRD1 was downregulated in both surgical patients and mice exposed to mechanical ventilation. Prophylactic administration of dopamine or DRD1 agonist attenuated mechanical stretch-induced lung endothelial barrier dysfunction and lung injury. By contrast, pulmonary knockdown or global knockout of DRD1 exacerbated these effects. Prophylactic administration of dopamine attenuated mechanical stretch-induced α-tubulin deacetylation and subsequent endothelial hyperpermeability through DRD1 signaling. We identified that cyclic stretch-induced glycogen-synthase-kinase-3ß activation led to phosphorylation and activation of histone deacetylase 6 (HDAC6), which resulted in deacetylation of α-tubulin. Upon activation, DRD1 signaling attenuated mechanical stretch-induced α-tubulin deacetylation and subsequent lung endothelial barrier dysfunction through cAMP/exchange protein activated by cAMP (EPAC)-mediated inactivation of HDAC6. Conclusions: This work identifies a novel protective role for DRD1 against mechanical stretch-induced lung endothelial barrier dysfunction and lung injury. Further study of the mechanisms involving DRD1 in the regulation of microtubule stability and interference with DRD1/cAMP/EPAC/HDAC6 signaling may provide insight into therapeutic approaches for VILI.

Upregulation of sphingosine kinase 1 contributes to ventilator-associated lung injury in a two-hit model.

Ventilator­associated lung injury (VALI) remains a significant medical problem in intensive care units. The present study aimed to investigate the role of sphingosine kinase 1 (SPHK1) in VALI using a two­hit model and explore the potential underlying molecular mechanism. Mice were divided into five groups: i) Non­ventilated group; ii) non­ventilated + lipopolysaccharide (LPS) group; iii) ventilated group; iv) ventilated + LPS group; and v) ventilated + LPS + SPHK1 inhibitor group. Mice were administered LPS (1 mg/kg) via an intraperitoneal injection. After 12 h, the mice were anesthetized and connected to a ventilator (10 ml/kg at 150 breaths/min) for 4 h. SPHK1 inhibitor (50 mg/kg) was injected intraperitoneally 1 h prior to ventilation. Mouse lung vascular endothelial cells were treated with LPS and SPHK1 inhibitor, and then subjected to cyclic stretch for 4 h. The present results suggested that the expression of SPHK1 and sphingosine 1 phosphate was upregulated in the two­hit model of VALI; SPHK1 inhibitor could attenuate VALI in the two­hit model as observed by hematoxylin and eosin staining, and affected the cell count and the protein content levels in the bronchoalveolar lavage fluid. In addition, treatment with SPHK1 inhibitor reduced the wet­to­dry ratio of the lungs and suppressed Evans blue dye leakage into the lung tissue. Furthermore, SPHK1 inhibitor exhibited protective effects on the two­hit model of VALI by inhibiting the Ras homolog family member a­mediated phosphorylation of myosin phosphatase target subunit 1 (MYPT­1) and endothelial hyperpermeability. Additionally, mice were divided into five additional groups: i) Non­ventilated group; ii) non­ventilated + LPS group; iii) ventilated group; iv) ventilated + LPS group; and v) ventilated + LPS + Rho­associated coiled­coil forming protein kinase (ROCK)1 inhibitor group. ROCK1 inhibitor (10 mg/kg) was injected intraperitoneally 1 h prior to ventilation. The present results suggested that ROCK1 inhibitor could attenuate mechanical stretch­induced lung endothelial injury and the phosphorylation of MYPT­1 in vivo and in vitro. Collectively, the present findings indicated that upregulation of SPHK1 may contribute to VALI in a two­hit model.

Downregulation of R-Spondin1 Contributes to Mechanical Stretch-Induced Lung Injury.

OBJECTIVES: The R-spondin family attenuates tissue damage via tightening endothelium and preventing vascular leakage. This study aims to investigate whether R-spondins protect against mechanical stretch-induced endothelial dysfunction and lung injury and to reveal the underlying mechanisms. DESIGN: Randomized controlled study. SETTING: University research laboratory. SUBJECTS: Patients scheduled to undergo surgery with mechanical ventilation support. Adult male Institute of Cancer Research mice. Primary cultured mouse lung vascular endothelial cells. INTERVENTIONS: Patients underwent a surgical procedure with mechanical ventilation support of 3 hours or more. Mice were subjected to mechanical ventilation (6 or 30 mL/kg) for 0.5-4 hours. Another group of mice were intraperitoneally injected with 1 mg/kg lipopolysaccharide, and 12 hours later subjected to mechanical ventilation (10 mL/kg) for 4 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 4 hours. MEASUREMENTS AND MAIN RESULTS: R-spondin1 were downregulated in both surgical patients and experimental animals exposed to mechanical ventilation. Intratracheal instillation of R-spondin1 attenuated, whereas knockdown of pulmonary R-spondin1 exacerbated ventilator-induced lung injury and mechanical stretch-induced lung vascular endothelial cell apoptosis. The antiapoptotic effect of R-spondin1 was mediated through the leucine-rich repeat containing G-protein coupled receptor 5 in cyclic stretched mouse lung vascular endothelial cells. We identified apoptosis-stimulating protein of p53 2 as the intracellular signaling protein interacted with leucine-rich repeat containing G-protein coupled receptor 5. R-spondin1 treatment decreased the interaction of apoptosis-stimulating protein of p53 2 with p53 while increased the binding of apoptosis-stimulating protein of p53 2 to leucine-rich repeat containing G-protein coupled receptor 5, therefore resulting in inactivation of p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. CONCLUSIONS: Mechanical ventilation leads to down-regulation of R-spondin1. R-spondin1 may enhance the interaction of leucine-rich repeat containing G-protein coupled receptor 5 and apoptosis-stimulating protein of p53 2, thus inactivating p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. R-spondin1 may have clinical benefit in alleviating mechanical ventilator-induced lung injury.

Magnesium transporter protein solute carrier family 41 member 1 suppresses human pancreatic ductal adenocarcinoma through magnesium-dependent Akt/mTOR inhibition and bax-associated mitochondrial apoptosis.

The aim of this study was to identify the function of the Mg2+ transporter protein solute carrier family 41 member 1 SLC41A1 in pancreatic ductal adenocarcinoma and the underlying mechanisms. A total of 27 solute carrier proteins were differentially expressed in pancreatic ductal adenocarcinoma. Three of these proteins were correlated with clinical outcomes in patients, among which SLC41A1 was downregulated in tumour. Overexpression of SLC41A1 suppressed orthotopic tumour growth in a mouse model and reduced the cell proliferation, colony formation, and invasiveness of KP3 and Panc-1 cells, which may have been associated with the increased population of apoptotic-prone cells. Overexpression of SLC41A1 reduced the mitochondrial membrane potential, induced Bax while suppressed Bcl-2 expression. Suppression of Bax abrogated the tumour-suppressive effects of SLC41A1. Furthermore, overexpression of SLC41A1 promoted Mg2+ efflux and suppressed Akt/mTOR activity, which is the upstream regulator of Bax and Bcl-2. An increase in Akt activity and supplementation with Mg2+ abolished SLC41A1-induced tumour suppression. The results of this study suggest that SLC41A1 may be a potential target for the treatment of pancreatic ductal adenocarcinoma.