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.

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.

Role of Epigenetics in the Pathogenesis, Treatment, Prediction, and Cellular Transformation of Asthma.

Asthma is a mysterious disease with heterogeneity in etiology, pathogenesis, and clinical phenotypes. Although ongoing studies have provided a better understanding of asthma, its natural history, progression, pathogenesis, diversified phenotypes, and even the exact epigenetic linkage between childhood asthma and adult-onset/old age asthma remain elusive in many aspects. Asthma heritability has been established through genetic studies, but genetics is not the only influencing factor in asthma. The increasing incidence and some unsolved queries suggest that there may be other elements related to asthma heredity. Epigenetic mechanisms link genetic and environmental factors with developmental trajectories in asthma. This review provides an overview of asthma epigenetics and its components, including several epigenetic studies on asthma, and discusses the epigenetic linkage between childhood asthma and adult-onset/old age asthma. Studies involving asthma epigenetics present valuable novel approaches to solve issues related to asthma. Asthma epigenetic research guides us towards gene therapy and personalized T cell therapy, directs the discovery of new therapeutic agents, predicts long-term outcomes in severe cases, and is also involved in the cellular transformation of childhood asthma to adult-onset/old age asthma.

Liver-specific over-expression of Cripto-1 in transgenic mice promotes hepatocyte proliferation and deregulated expression of hepatocarcinogenesis-related genes and signaling pathways.

In this study, we investigated the role of embryonic gene Cripto-1 (CR-1) in hepatocellular carcinoma (HCC) using hepatocyte-specific CR-1-overexpressing transgenic mice. The expression of truncated 1.7-kb CR-1 transcript (SF-CR-1) was significantly higher than the full-length 2.0-kb CR-1 transcript (FL-CR-1) in a majority of HCC tissues and cell lines. Moreover, CR-1 mRNA and protein levels were significantly higher in HCC tissues than adjacent normal liver tissues. Hepatocyte-specific over-expression of CR-1 in transgenic mice enhanced hepatocyte proliferation after 2/3 partial hepatectomy (2/3 PHx). CR-1 over-expression significantly increased in vivo xenograft tumor growth of HCC cells in nude mice and in vitro HCC cell proliferation, migration, and invasion. CR-1 over-expression in the transgenic mouse livers deregulated HCC-related signaling pathways such as AKT, Wnt/ß-catenin, Stat3, MAPK/ERK, JNK, TGF-ß and Notch, as well as expression of HCC-related genes such as CD5L, S100A8, S100A9, Timd4, Orm2, Orm3, PDK4, DMBT1, G0S2, Plk2, Plk3, Gsta1 and Gsta2. However, histological signs of precancerous lesions, hepatocyte dysplasia or HCC formation were not observed in the livers of 3-, 6- or 8-month-old hepatocyte-specific CR-1-overexpressing transgenic mice. These findings demonstrate that liver-specific CR-1 overexpression in transgenic mice deregulates signaling pathways and genes associated with HCC.

The Yunnan national medicine Maytenus compound inhibits the proliferation of hepatocellular carcinoma (HCC) by suppressing the activation of the EGFR-PI3K-AKT signaling pathway.

Objective: To investigate the effects of Maytenus compound on the proliferation of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to explore the underlying mechanism. Methods: The half maximal inhibitory concentration (IC50) values of Maytenus compound in HepG2 and BEL-7402 cells were determined by the MTS assay. HepG2 and BEL-7402 cells were treated with different concentrations of Maytenus compound. MTS assays, colony formation assays and cell cycle analyses were performed to clarify the inhibitory effect of Maytenus compound on the proliferation of HepG2 and BEL-7402 cells in vitro. After subcutaneous injection of HepG2 cells, nude mice were randomly divided into a vehicle control group and a drug intervention group, which were intragastrically administered ddH2O or Maytenus compound, respectively. The inhibitory effect of Maytenus compound on the proliferation of HepG2 cells in vivo was analyzed using subcutaneous tumor growth curves, tumor weight, the tumor growth inhibition rate and the immunohistochemical detection of BrdU-labeled cells in S phase. The organ toxicity of Maytenus compound was initially evaluated by comparing the weight difference and organ index of the two groups of nude mice. The main proteins in the EGFR-PI3K-AKT signaling pathway were detected by Western blot after Maytenus compound intervention in vivo and in vitro. Results: Maytenus compound showed favorable antiproliferation activity against HepG2 and BEL-7402 cells with IC50 values of 79.42±11.71 µg/mL and 78.48±8.87 µg/mL, respectively. MTS assays, colony formation assays and cell cycle analyses showed that Maytenus compound at different concentration gradients within the IC50 concentration range significantly suppressed the proliferation of HepG2 and BEL-7402 cells in vitro and inhibited cell cycle progression from G1 to S phase. Additionally, Maytenus compound, at an oral dose of 2.45 g/kg, dramatically inhibited, without obvious organ toxicity, the proliferation of subcutaneous tumors formed by HepG2 cells in nude mice. In addition, the tumor growth inhibition rate for Maytenus compound was 66.94%. Furthermore, Maytenus compound inhibited the proliferation of liver orthotopic transplantation tumors in nude mice. Western blot analysis showed that Maytenus compound significantly downregulated the expression of p-EGFR, p-PI3K, and p-AKT and upregulated the expression of p-FOXO3a, p27, and p21 in vivo and in vitro. Conclusion: Maytenus compound significantly inhibited the proliferation of HCC cells in vitro and in vivo. The downregulation of the EGFR-PI3K-AKT signaling pathway and subsequent inhibition of cell cycle progression from G1 to S phase is one of the possible mechanisms. Maytenus compound has a high tumor growth inhibition rate and has no obvious organ toxicity, which may make it a potential anti-HCC drug, but the results from this study need to be confirmed by further clinical trials in HCC patients.

Role of Sex Hormones at Different Physiobiological Conditions and Therapeutic Potential in MBD2 Mediated Severe Asthma.

Sex hormone has become a "hot topic" to evaluate the hormonal therapeutic potential in severe asthma. Th17 cell is one of the main influencing factors involved in the pathogenesis of severe asthma, hence also called as kernel of severe asthma, and Th17 subtype of non-T2 asthma is less responsive (resistance) to inhaled corticosteroid (ICS), so severe in nature. Methyl-CpG binding domain protein 2 (MBD2) is overexpressed and regulates the Th17 differentiation, showing the possibility of therapeutic target in treating Th17 mediated severe asthma. Sex hormone fluctuates at the different physiobiological conditions of the human body and affects the asthma pathobiology showing its role in asthma prevalence, severity, remission, and therapy. This review briefly overviews the sex hormones, their influence in asthma at the different physiobiological conditions of human body, and MBD2 severe asthma connection with the possible therapeutic potential of sex steroids in MBD2 mediated Th17 predominant severe asthma. Male sex hormone tends to show a beneficial effect and possibly downregulates the expression of Th17 cells via regulating MBD2 through a mechanism distinct from corticosteroid treatment and guides us towards discovery of new therapeutic agent, reduces the asthma-related complications, and promotes long-term survival by lowering the risk of therapy-resistant issues of old age severe asthma.

Alanylglutamine Relieved Asthma Symptoms by Regulating Gut Microbiota and the Derived Metabolites in Mice.

OBJECTIVE: Allergic asthma is a chronic inflammatory disease, which seriously affects the life quality of patients, especially children. Alanylglutamine is a nutritional supplement with potential protective and anti-inflammatory effects, but its function in allergic asthma remains elusive. In this study, we focused on the investigations of the roles and functional mechanism of Alanylglutamine in asthma. METHODS: Ovalbumin (OVA) induction was utilized to establish a mouse asthma model. 16S rDNA sequencing was performed to compare the diversity of intestinal microorganisms under different treatments. Gas chromatography was utilized to screen the intestinal microbe-short-chain fatty acids in the stool. The lung tissue was extracted to determine signaling pathways, including AMPK, NF-κB, mTOR, STAT3, IKKß, TGF-ß, and IL-1ß through Western blot or RT-qPCR. RESULTS: It was observed that Alanylglutamine reduced the cytokine in OVA-induced allergic asthma mice. H&E staining showed obvious pneumonia symptoms in the asthma group, while Alanylglutamine alleviated the inflammatory infiltration. Alanylglutamine reversed gut microbiota compositions in OVA-induced allergic asthma mice and enhanced the butyric acid level. The protective role of Alanylglutamine may be associated with the gut microbiota-butyric acid-GPR43 pathway in asthma mice. In contrast to the OVA group, Alanylglutamine activated the protein expression of P-AMPK/AMPK and inhibited the protein expression of P-mTOR/mTOR, P-P65/P65, P-STAT3/STAT3, P-IKKß/IKKß, TGF-ß, and IL-1ß, with similar effects from butyric acid. CONCLUSION: The results indicated that Alanylglutamine might be beneficial for asthma, and its effect was achieved through the regulation on microbiota and the derived metabolites. The therapeutic effects might be associated with AMPK, NF-κB, mTOR, and STAT3 signaling pathways. These findings will help identify effective therapeutic direction to alleviate allergic inflammation of the lungs and airways.

The effect of pollutional haze on pulmonary function.

Detrimental health effects of atmospheric exposure to ambient particulate matter (PM) have been investigated in numerous studies. Exposure to pollutional haze, the carrier of air pollutants such as PM and nitrogen dioxide (NO2) has been linked to lung and cardiovascular disease, resulting increases in both hospital admissions and mortality. This review focuses on the constituents of pollutional haze and its effects on pulmonary function. The article presents the available information and seeks to correlate pollutional haze and pulmonary function.

IL-32 was involved in cigarette smoke-induced pulmonary inflammation in COPD.

PURPOSE: Previous study has proven the overexpression of interleukin 32 (IL-32) in lungs with chronic obstructive pulmonary disease (COPD). But the soluble IL-32 levels and the role of IL-32 in smokers and COPD are still unclear. METHODS: In this study, we enrolled 133 subjects who were divided into three groups: nonsmokers, control smokers and smokers with COPD. We detected the IL-32 levels in serum and induced sputum of all subjects. The pulmonary function, PaO2 and smoking exposure index were also collected. Moreover, macrophages were isolated and stimulated by cigarette smoke extraction (CSE). A special siRNA was used to suppress the IL-32 expression. RESULTS: There was no significant difference in IL-32 serum levels among the three groups. The IL-32 levels of induced sputum in COPD patients were markedly higher than control smokers and nonsmokers. The IL-32 levels in induced sputum of COPD patients were negatively correlated with forced expiratory volume (FEV1)/forced vital capacity and FEV1%. Moreover, a low concentration CSE could stimulate IL-32 expression and promote the release of several inflammatory factors (such as IL-6 and tumor necrosis factor-α). A special siRNA could significantly suppress the release of these inflammatory factors. CONCLUSIONS: This study revealed the critical role of IL-32 in pulmonary inflammation of COPD and smoker-associated diseases.

Oxidized low-density lipoprotein, OXPAPC, corrects defects in maturation and cytokine secretion of peripheral blood dendritic cells from sepsis patients.

OBJECTIVE: To investigate the expression differences in maturation and cytokine production of dendritic cells (DCs) from sepsis patients and the effect of oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OXPAPC) on DCs phenotypes. METHODS: Peripheral blood mononuclear cells from 50 sepsis patients and 50 controls were cultured in the presence of GM-CSF, IL-4 and TNF-α to induce DCs maturation. DCs from sepsis patients were also treated with three different concentrations of OXPAPC. Cells were characterized with optical and electron microscopy, FACS analysis for CD1α, HLA-DR and CD86 on cell surface and ELISA analysis of IL-12p70 in the supernatant. RESULTS: DCs from sepsis patients had smaller cell bodies and nucleus and had almost no surface projection. DCs had similar CD1α expression in sepsis patients (86.37 ± 17.24) and controls (88.58 ± 10.05). HLA-DR expression was dramatically reduced in sepsis patients (2.74 ± 5.15) compared to controls (198.35 ± 12.04). Similarly, CD86 expression was also drastically lower in sepsis patients (14.72 ± 4.83) than controls (154.56 ± 11.56). Furthermore, OXPAPC treatment of DCs from sepsis patients increased cell surface projection, HLA-DR and CD86 surface expression and IL-12p70 secretion in a dose-dependent manner. With 40 µg/ml of OXPAPC, DCs of sepsis patients have similar phenotypes observed in healthy controls. CONCLUSION: DCs from sepsis patients are defective in maturation and cytokine secretion and these defects can be corrected by OXPAPC treatment.

In vitro study on blocking mTOR signaling pathway in EGFR-TKI resistance NSCLC.

OBJECTIVE: To investigate the effect and mechanism of inhibitor everolimus on EGFR-TKI resistance NSCLC. METHODS: MTT assay was used to detect proliferation of human non-small cell lung cancer cell line A549. Flow cytometry was used to detect the changes of apoptosis and cycle distribution in each group after 24 h and 48 h. RT-PCR was used to detect the changes of PTEN and 4EBP1 expression levels after 48 h of monotherapy and combination therapy. RESULTS: MTT assay showed that everolimus had dose-dependent inhibition against growth of A549 cells. Flow cytometry showed when everolimus could induce apoptosis and induce G0/G1 phase cell cycle arrest, which was time-dependent (P<0.05). RT-PCR showed everolimus could increase PTEN and 4EBP1 expression. CONCLUSIONS: mTOR inhibitor everolimus has an inhibitory effect on EGFR-TKI resistant NSCLC, which cannot reverse the resistance effect of EGFR-TKI resistant cell line A549. The relationship between EGFR/AKT signaling pathway and the mTOR signaling pathway and the mechanism in non-small cell lung cancer need further study.

Expression and methylation of mitochondrial transcription factor a in chronic obstructive pulmonary disease patients with lung cancer.

BACKGROUND: Apoptosis plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), and this process can be regulated by mitochondrial transcription factor A (mtTFA). Epigenetics is involved in the regulation and modification of the genes involved in lung cancer and COPD. In this study, we determined the expression of mtTFA and its methylation levels in the COPD patients with lung cancer. METHODS: Twenty-one squamous cell lung cancer patients, 11 with COPD and 10 without COPD, undergoing pneumonectomy were enrolled. The apoptotic index (AI) of pulmonary vascular endothelial cells was analyzed by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. The expression of mtTFA mRNA and protein was measured using PCR, immunohistochemistry and Western-blot. Methylation of the mtTFA promoter was detected using bisulfite sequencing PCR. RESULTS: Compared to the non-COPD group, the AI was higher, and expression of mtTFA mRNA and protein was lower in the COPD group (P<0.001). Expression of the mtTFA protein was positively correlated with FEV1/Pre (r = 0.892, P<0.001), and negatively correlated with AI (r = -0.749, P<0.001) and smoke index (r = -0.763, P<0.001). Percentage of mtTFA promoter methylation in the COPD patients was significantly higher compared to the non-COPD patients (P<0.05). CONCLUSION: These results suggest that the expression of mtTFA mRNA and protein is down-regulated in the lung tissue from the COPD patients with squamous cell lung cancer, and the level of mtTFA protein is related to apoptosis of pulmonary vascular endothelial cells. Aberrant mtTFA methylation may also play an important role in the pathogenesis of COPD.

Decreased and dysfunctional circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease.

BACKGROUND: It has been widely demonstrated that endothelial progenitor cells are involved in several diseases and that they have therapeutic implications. In order to define the altered pulmonary vascular homeostasis in chronic obstructive pulmonary disease, we sought to observe the level and functions of circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease. METHODS: The total study population included 20 patients with chronic obstructive pulmonary disease and 20 control subjects. The number of circulating endothelial progenitor cells (CD34(+)/CD133(+)/VEGFR-2(+) cells) was counted by flow cytometry. Circulating endothelial progenitor cells were also cultured in vitro and characterized by uptake of DiIacLDL, combining with UEA-I, and expression of von Willebrand factor and endothelial nitric oxide synthase. Adhesion, proliferation, production of nitric oxide, and expression of endothelial nitric oxide synthase and phosphorylated-endothelial nitric oxide synthase were detected to determine functions of circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease. RESULTS: The number of circulating endothelial progenitor cells in the chronic obstructive pulmonary disease group was lower than in the control group: (0.54 ± 0.16)% vs. (1.15 ± 0.57)%, P < 0.05. About 80% of adherent peripheral blood mononuclear cells cultured in vitro were double labeled with Dil-acLDL and UEA-1. The 92% and 91% of them were positive for von Willebrand factor and endothelial nitric oxide synthase, respectively. Compared with the control, there were significantly fewer adhering endothelial progenitor cells in chronic obstructive pulmonary disease patients: 18.7 ± 4.8/field vs. 45.0 ± 5.9/field, P < 0.05. The proliferation assay showed that the proliferative capacity of circulating endothelial progenitor cells from chronic obstructive pulmonary disease patients was significantly impaired: 0.135 ± 0.038 vs. 0.224 ± 0.042, P < 0.05. Furthermore, nitric oxide synthase (112.06 ± 10.00 vs. 135.41 ± 5.38, P < 0.05), phosphorylated endothelial nitric oxide synthase protein expression (88.89 ± 4.98 vs. 117.98 ± 16.49, P < 0.05) and nitric oxide production ((25.11 ± 5.27) µmol/L vs. (37.72 ± 7.10) µmol/L, P < 0.05) were remarkably lower in endothelial cells from the chronic obstructive pulmonary disease group than the control. CONCLUSION: Circulating endothelial progenitor cells were decreased and functionally impaired in patients with chronic obstructive pulmonary disease.

Open resection for lung cancer in right upper lobe.

An open radical surgery for lung cancer of the right upper lobe is performed under suitable conditions in this case. According to the actual conditions, the horizontal fissure is made a "tunnel" dissociation during the operation to fully expose hilar structures (artery, vein, and bronchus). Since intraoperative frozen section diagnosis shows malignant result, lymph nodes are dissected. Hemostasis, protection of the important peripheral organs and standard postoperative placement of drainage tube should be noted. The observability of this surgery is the clear exposure and brief operation.

Effects and mechanism of arsenic trioxide on reversing the asthma pathologies including Th17-IL-17 axis in a mouse model.

In traditional Chinese medicine, arsenous compounds, including arsenic trioxide (ATO), are often used to treat many diseases, which are safe and effective. Recently, studies have indicated that Th17- IL-17 involved in the pathogenesis and development of asthma. The goal of this study was to investigate the effect and mechanism of ATO on asthma, especially the Th17- IL-17 axis.We used oval bumin (OVA)-immunized mice as a model for asthma and treated mice with ATO or dexamethasone. The mice were then monitored airway responsiveness, airway inflammation, mucus production, IL-17 levels in BALF and the positive rate of Th17 cells. In vitro, CD4+ T cells from splenic cell suspensions were separated and purified. We measured the expression of IL-17 and caspase-12 protein in purified CD4+ T cells, and detected IL-17 levels in CD4+ T lymphocyte culture solution with or without ATO. Moreover, apoptosis, mitochondrial membrane potential, cytosolic calcium were analyzed. We found that ATO could reduce airway responsiveness, airway inflammation, mucus hyperplasia, the expression of IL-17 in BALF and the positive rate of Th17 cells at a level comparable to treatment with DXM. In vitro data suggested that ATO can induce CD4+ T cells apoptosis, cause mitochondrial dysfunction, Ca2+ overload and promote caspase-12 activation. Our study suggested that ATO had potential medical value for the treatment of human asthma..

ß-arrestin2 stimulates interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model.

Allergic asthma is a complex and chronic inflammatory airway disease. Interleukin-17 is a pro-inflammatory cytokine which plays critical role in the pathogenesis of allergic asthma. It has been reported that ß-arrestin2 regulated the development of allergic asthma at a proximal step in the inflammatory cascade. In this study, the influence of ß-arrestin2 on Interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model was investigated. Splenic CD4+ T lymphocytes from wild-type mice and those from a murine asthma model were purified. CD4+ T lymphocytes from a murine asthma model were transfected with siRNAs targeting the ß-arrestin2 or were pretreated with the ERK1/2 inhibitor, PD98059. After stimulation, the protein expression of ß-arrestin2、phosphorylated-ERK1/2 and IL-17 were detection by Western blot; the mRNA expression of IL-17 were detected by real-time PCR; the accumulation of IL-17 in supernatants were detected by ELISA. We found that ß-arrestin2、phosphorylated-ERK1/2 and IL-17 expression in CD4+ T lymphocytes from a murine asthma model were increased compared with those from wild-type mice (p < 0.01). Treatment of CD4+ T lymphocytes with siRNAs targeting the ß-arrestin2 down-regulated phosphorylated- ERK 1/2 and IL-17 expression (p < 0.01). PD98059 decreased IL-17 production and expression in CD4+ T lymphocytes in a murine asthma model (p < 0.05). We conclude that ß-arrestin2 stimulated IL-17 production and expression of CD4+ T lymphocytes in a murine asthma model. The effect was partly mediated by ERK 1/2 activation. Targeting ß-arrestin2 biological activity could be a valid therapeutic approach for the treatment of allergic asthma.

[The expression of prostacyclin in lungs from patients with chronic obstructive pulmonary disease].

OBJECTIVE: to investigate the expression of prostacyclin in lungs from patients with chronic obstructive pulmonary disease (COPD). METHODS: the lung tissues were obtained from 12 patients with COPD and 10 patients without COPD. The apoptosis of pulmonary vascular endothelial cells was analyzed quantitatively using TdT-mediated dUTP nick end labeling (TUNEL). The expression of PGI2-S protein was assessed by immunohistochemistry of paraffin-embedded tissues. 6-keto Prostaglandin F1a, the stable metabolite of PGI2, was measured by enzyme-linked immunosorbent assay in whole-lung tissue extracts. The data distributed normally were expressed as x(-) +/- s, and the independent-samples t test was used for comparison of means. RESULTS: the apoptotic index (AI) of endothelial cells of medium and small-sized vessels in patients with COPD group [(12.9 +/- 2.0)%, (11.4 +/- 1.4)%] were significantly higher than that of patients without COPD [(6.1 +/- 1.2)%, (5.9 +/- 0.4)%]. In patients without COPD, PGI2-S protein expression in medium vessels and small-sized vessels was (95.4 +/- 2.1)% and (82.3 +/- 7.4)% respectively, and the value was (95.5 +/- 2.2)% in airway epithelial cells. In patients with COPD, PGI2-S expression in medium vessels and small-sized vessels decreased remarkably [(55.2 +/- 9.8)% and (42.3 +/- 5.1)% respectively], and exhibited a marked reduction in airway epithelial cells (31.8 +/- 5.2)%. The level of 6-keto Prostaglandin F1a was significantly lower in patients with COPD (2.6 +/- 0.4)microg/L compared with patients without COPD (16.2 +/- 2.8)microg/L. CONCLUSION: abnormal apoptosis exists in pulmonary vascular endothelial cells in patients with COPD. In patients with COPD, the expression of PGI2-S and the level of 6-keto Prostaglandin F1a in lung tissues were decreased. The results suggest that abnormal apoptosis, reduction of PGI2-S expression and PGI2 may be important histological markers for pulmonary endothelial cell dysfunction in COPD and may be involved in the pathogenesis of the disease.

[Effect of cyclooxygenase-2 on vascular endothelial cell apoptosis induced by cigarette smoke extract].

OBJECTIVE: To explore the effect of cyclooxygenase-2 on vascular endothelial cell apoptosis induced by cigarette smoke extract. METHODS: Human vascular endothelial cells (ECV-304) were cultured in vitro, and those at the exponential growth phase were studied in experiments. The experiment was completed through 3 steps: (1) ECV-304 cells were cultured with 0.0%, 0.5%, 1.0% and 5.0% CSE for 12 h. (2) ECV-304 cells were exposed to 5.0% CSE for 0, 3, 6, 9, 12 and 24 h. (3) Endothelial cells were treated by 5% CSE, together with different concentrations of selective COX-2 inhibitor celecoxib (0.0, 2.5, 5.0, 10.0, 20.0, 50.0 micromol/L concentrations) for 9 h. The cell apoptosis rate was tested by Hoechst staining and flow cytometry methods, and the expression of COX-2 protein by immunocytochemistry and Western blotting. RESULTS: CSE induced ECV-304 cell apoptosis and COX-2 expression in a dose-dependent manner. The apoptosis rate of ECV-304 cells with 5.0% CSE was the highest (5.40+/-0.39)%. CSE- induced COX-2 expression reached the highest level with 5.0% CSE (206.1+/-15.5), the differences being significant (F=90.03, 159.94, all P<0.05). Furthermore CSE induced both apoptosis rate and COX-2 expression time-dependently, with the apoptosis rate achieving the peak after 24 h (8.87+/-0.41)%, while COX-2 expression reached the highest level at 9 h. The selective COX-2 inhibitor celecoxib inhibited COX-2 protein expression partially and augmented cell apoptosis induced by CSE. CONCLUSIONS: CSE induces endothelial cell apoptosis and increases the expression of COX-2 protein in vascular endothelial cells. Celecoxib, the selective COX-2 inhibitor, reduces the expression of COX-2 protein and promotes cell apoptosis induced by CSE in vascular endothelial cells. COX-2 may play an important role in protecting development of CSE-associated apoptosis of endothelial cells.

[Effect of rosiglitazone on the expression of T-bet/GATA-3 in T lymphocytes in patients with acute asthma].

OBJECTIVE: To investigate the effect of rosiglitazone on the expression of T-bet/GATA-3 in peripheral blood T lymphocytes and cytokine derived from Th1/Th2 in patients with acute asthma and to compare its mechanism with that of dexamethasone (DXM). METHODS: Peripheral blood T lymphocytes from 10 patients with acute asthma (A group, A) and 10 healthy volunteers (B group, B) were isolated, purificated and cultured, and were divided into 2 groups (3 h and 24 h) based on the treatment time, and each group was further divided, on the basis of the treatment given, into 3 sub-groups respectively: control group (A(1) and B(1)), rosiglitazone treated group (A(2) and B(2)) and DXM treated group (A(3) and B(3)). The levels of IFN-gamma and IL-4 in the culture supernatant were detected by Enzyme-linked immunoadsorbent assay (ELISA) and the expression levels of T-bet mRNA/GATA-3 mRNA in T lymphocytes was detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). RESULTS: The levels of INF-gamma and INF-gamma/IL-4 in the culture supernatant of T lymphocytes and T-bet mRNA and T-bet mRNA/GATA-3 mRNA in T lymphocytes from the A at 3 h and 24 h were 357 +/- 31, 783 +/- 47, 5.5 +/- 1.0, 8.4 +/- 1.5, 18.7 +/- 3.7, 11.9 +/- 2.9, 1.20 +/- 0.11, 0.290 +/- 0.020, and those from the B at 3 h and 24 h were 659 +/- 41, 1394 +/- 120, 11.5 +/- 3.0, 17.4 +/- 4.0, 29.0 +/- 4.0, 18.9 +/- 4.0, 3.82 +/- 0.81, 0.870 +/- 0.090, the differences were significant between these groups (t = 18.59, 5.95, 14.87, 6.25, 6.06, 4.63, 10.23, 18.67, all P < 0.05, respectively). The levels of IL-4 in the culture supernatant of T lymphocytes and GATA-3 mRNA in T lymphocytes from the A at 3 h and 24 h were 65 +/- 6, 96 +/- 9, 16.4 +/- 4.2, 41 +/- 6, and those from the B at 3 h and 24 h were 57 +/- 5, 83 +/- 7, 7.8 +/- 2.2, 23 +/- 4, the differences were significant between these groups (t = 3.19, 3.90, 5.77, 7.76, all P < 0.05, respectively). The levels of IFN-gamma, IL-4 and IFN-gamma/IL-4 in the culture supernatant of T lymphocytes from the A and B at 3 h were 357 +/- 31, 65 +/- 6, 5.5 +/- 1.0, 659 +/- 41, 57 +/- 5, 11.5 +/- 3.0, and those from the A and B at 24 h were 783 +/- 47, 96 +/- 9, 8.4 +/- 1.5, 1394 +/- 120, 83 +/- 7, 17.4 +/- 4.0, The differences were significant between these groups (t = 23.8, 9.48, 5.03, 18.21, 9.01, 3.53, all P < 0.05, respectively). The levels of T-bet mRNA and T-bet mRNA/GATA-3 mRNA in T lymphocytes from the A and B at 3 h were 18.7 +/- 3.7, 1.20 +/- 0.11, 29.0 +/- 4.0, 3.82 +/- 0.81, and those from the A and B at 24 h were 11.9 +/- 2.9, 0.290 +/- 0.020, 18.9 +/- 4.0, 0.870 +/- 0.090, the differences were significant between these groups (t = 4.62, 5.66, 29.67, 11.5, all P < 0.05, respectively). The levels of GATA-3 mRNA in T lymphocytes from the A and B at 3 h were 16.4 +/- 4.2, 7.8 +/- 2.2, it was lower than those from the A and B at 24 h (41 +/- 6, 23 +/- 4, t = 10.70, 10.11, all P < 0.05, respectively). The levels of T-bet mRNA from the A showed a positive correlation with the level of IFN-gamma (r = 0.581, P < 0.05), and a negative correlation with the level of IL-4 (r = -0.493, P < 0.05); The levels of GATA-3 mRNA from the A showed a negative correlation with the level of IFN-gamma (r = -0.501, P < 0.05), and a positive correlation with the level of IL-4 (r = 0.579, P < 0.05); The ratio of T-bet mRNA to GATA-3 mRNA from the A showed a positive correlation with the ratio of IFN-gamma to IL-4 (r = 0.696, P < 0.05). CONCLUSION: Rosiglitazone could regulate the balance of IFN-gamma and IL-4 through effect on the expression of T-bet mRNA and GATA-3 mRNA.

Statins may ameliorate pulmonary hypertension via RhoA/Rho-kinase signaling pathway.

Statins are the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that function as potent inhibitors of cholesterol biosynthesis and have been used for many years for the treatment of hypercholesterolemia. However, accumulating experimental and clinical studies have revealed that the health benefits associated with statins treatment, particularly those conferred on the cardiovascular system, were the cholesterol-independent. Because statins inhibit an early step in the cholesterol biosynthetic pathway, they also inhibit the synthesis of isoprenoids such as farnesylpyrophosphate and geranylgeranylpyrophosphate, which are important postranslational lipid attachments for intracellular signaling molecules such as the Rho GTPases. The isoprenylation of Rho is a prerequisite for Rho activation, facilitating its interaction with the plasma membrane, undergoing GDP-GTP exchange and be activated. Inhibition of RhoA geranylgeranylation by statins decreases membrane GTP-bound active RhoA and subsequent Rho-kinase activity. Activated RhoA via its downstream effector Rho-kinase is involved in a wide range of cellular functions, such as cell migration, proliferation and apoptosis. Recently, rising evidences suggested that RhoA/Rho-kinase pathway was essentially involved in various models of pulmonary hypertension and statins effectively ameliorated pulmonary hypertension. Based on this findings, we hypothesis that statins attenuate pulmonary hypertension via RhoA/Rho-kinase signaling pathway in vivo.