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.

CHOP overexpression sensitizes human non-small cell lung cancer cells to cisplatin treatment by Bcl-2/JNK pathway.

C/EBP homologous protein (CHOP), a 29 kDa cellular protein, plays a role in regulating tumor proliferation, differentiation, metabolism, cell death, and in tumor resistance to chemotherapy. Non-small cell lung cancer (NSCLC) is a tumor of the respiratory system and drug resistance is prevalent among NSCLC clinical cell cultures. Herein, our study elucidated the effect of CHOP on NSCLC cells with cisplatin resistance and its mechanism. In a NSCLC cell line with cisplatin-resistance, CHOP expression was decreased, compared with A549 cells. Overexpression of CHOP decreased the cell viability and enhanced cell apoptosis in the cells treated with cisplatin. Expression of CHOP also inhibited the cell proliferation and metastasis. CHOP increased the therapeutic effect of cisplatin on NSCLC cells through the Bcl-2/JNK pathway. In summary, CHOP regulated cisplatin resistance in cells of NSCLC by promoting the expression of apoptotic proteins and inhibiting the Bcl-2/JNK signaling pathway, indicating the antitumor effects of CHOP.

Endoplasmic Reticulum Stress Induces HRD1 to Protect Alveolar Type II Epithelial Cells from Apoptosis Induced by Cigarette Smoke Extract.

BACKGROUND/AIMS: Cigarette smoking is a major risk factor of chronic obstructive pulmonary disease. This study aimed to examine the effects of cigarette smoke extract (CSE) on alveolar type II epithelial cells (AECII) and investigate the underlying mechanism. METHODS: Primary AECII were isolated from rat lung tissues and exposed to CSE. Apoptosis was detected by flow cytometry. Protein expression was detected by Western blot analysis. RESULTS: Primary rat AECII maintained morphological and physiological characteristic after 3 passages. CSE increased the expression of ER specific pro-apoptosis factors CHOP and caspase 12, and the phosphorylation of JNK in AECII. CSE activated ER stress signaling and increased the phosphorylation of PERK, eIF2α and IRE1. Furthermore, CSE induced the expression of Hrd1, a key factor of ER-associated degradation, in AECII. Knockdown of Hrd1 led to more than 2 fold increase of apoptosis, while overexpression of Hrd1 attenuated CSE induced apoptosis of AECII. CONCLUSIONS: Our results suggest that ER stress induces HRD1 to protect alveolar type II epithelial cells from apoptosis induced by CSE.

Iron-Catalyzed Direct Diazidation for a Broad Range of Olefins.

Reported herein is a new iron-catalyzed diastereoselective olefin diazidation reaction which occurs at room temperature (1-5 mol% of catalysts and d.r. values of up to >20:1). This method tolerates a broad range of both unfunctionalized and highly functionalized olefins, including those that are incompatible with existing methods. It also provides a convenient approach to vicinal primary diamines as well as other synthetically valuable nitrogen-containing building blocks which are difficult to obtain with alternative methods. Preliminary mechanistic studies suggest that the reaction may proceed through a new mechanistic pathway in which both Lewis acid activation and iron-enabled redox-catalysis are crucial for selective azido-group transfer.

Iron-Catalyzed Diastereoselective Intramolecular Olefin Aminobromination with Bromide Ion.

A new iron-catalyzed diastereoselective aminobromination method is reported for both internal and terminal olefins (yield up to 90% and dr up to >20:1). In this transformation, a functionalized hydroxylamine and bromide ion were used as the nitrogen and bromine source, respectively. This method is compatible with a broad range of olefins and provides a convenient approach to synthetically valuable vicinal bromo primary amines. Our studies suggest that both the diastereoselectivity and enantioselectivity for the olefin aminobromination can be controlled by iron catalysts.

Silver-mediated oxidative C-H/P-H functionalization: an efficient route for the synthesis of benzo[b]phosphole oxides.

A Ag-mediated C-H/P-H functionalization reaction of arylphosphine oxides with internal alkynes was described for the direct preparation of benzo[b]phosphole oxides with a high yield. An unusual aryl migration on the P-atom derived from a C-P bond cleavage and a new C-P bond formation was also observed and demonstrated to proceed via the radical process.

[The expression of hypoxia-inducible factor-1alpha and its hydroxylases in pulmonary arteries of patient with chronic obstructive pulmonary disease].

OBJECTIVE: To observe the expression of hypoxia-inducible factor-lalpha subunit (HIF-1alpha), HIF prolyl hydroxylase domain-containing protein(PHDs) and factor inhibiting HIF-1(FIH) in pulmonary arteries of patient with chronic obstructive pulmonary disease (COPD). METHODS: Pulmonary specimens were obtained from patients undergoing lobectomy for lung cancer, 12 had concurrent COPD (COPD group) and 14 without COPD (control group). The ratio of vascular wall area to total vascular area (WA%) and pulmonary artery media thickness (PAMT) was observed, and HIF-1alpha and its hydroxylases(PHD1, PHD2, PHD3, FIH) mRNA and protein were detected by in situ hybridization and immunohistochemistry respectively. RESULTS: WA% and PAMT of COPD patients(50 microm +/- 9 microm, 40% +/- 5%, were statistically different from those of the control subjects (39 microm +/- 6 microm, 31% +/- 4%, P < 0.01). Relative quantification of mRNA and protein levels (absorbance, A) showed that HIF-lalpha mRNA and protein levels in COPD group (0.230 +/- 0.036,0.275 +/- 0.039) were statistically higher than those of the control subjects (0.174 +/- 0.029, 0.102 +/- 0.015, P < 0.01 ), and that the protein level increased more markedly. PHD1 mRNA in COPD subjects (0.180 +/- 0.030) was comparable to that in control group (0.191 +/- 0.029, P > 0.05); PHD2 and PHD3 mRNA levels in COPD (0.245 +/- 0.044, 0.252 +/- 0.023) were significantly higher than those in control group(0.182 +/- 0.028, 0.127 +/- 0.017, P < 0.01). On the other hand, in COPD subjects PHD1 protein (0.104 +/- 0.015) was significantly lower(P < 0.01), whereas PHD2 protein (0.274 +/- 0.044) was significantly higher(P < 0.01) than those in control group(0.209 +/- 0.023, 0.219+/- 0.043). As for PHD3 protein, no significant changes were observed between the two groups (0.161+/- 0.023 in COPD, 0.146 +/- 0.021 in control, P > 0.05). FIH mRNA and protein both showed no differences between the two groups. Linear correlation analysis showed that HIF1alpha protein was positively correlated with WA%, PAMT, PHD2 mRNA and protein, PHD3 mRNA, and that HIF1alpha protein was negatively correlated with PHD1 protein. CONCLUSION: PHDs may be involved in the process of hypoxic pulmonary vascular remodeling in COPD via regulation of HIF-1alpha gene expression

Palladium-catalyzed 1,4-addition of diarylphosphines to α,ß-unsaturated aldehydes.

A highly stereoselective asymmetric 1,4-addition of diarylphosphines to α,ß-unsaturated aldehydes catalyzed by a bis(phosphine) pincer-Pd complex has been developed for the synthesis of chiral phosphines with excellent stereoselectivity (up to 98% ee) under mild conditions. The application of the current method to the synthesis of enantiopure bisphosphine and its pincer-Pd complex has also been demonstrated.

[Reciprocal regulation between hypoxia-inducible factor-1alpha and its prolyl hydroxylases in hypoxic pulmonary hypertension rats].

OBJECTIVE: To investigate the interaction between hypoxia-inducible factors-1alpha subunit (HIF-1alpha) and its three prolyl hydroxylases (PHD1, PHD2 and PHD3) during the development of rat hypoxic pulmonary hypertension. METHODS: Forty male SD rats were randomly divided into 5 groups and exposed to normoxia (C group) or exposed to hypoxia for 3, 7, 14 or 21 d (H(3), H(7), H(14), H(21) group), respectively. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were measured. Reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization were used to determine the expression of mRNA. Immunohistochemistry and Western blot were used to determine the expression of mRNA. RESULTS: The level of mPAP [(21.7 +/- 2.4) mm Hg, 1 mm Hg = 0.133 kPa], the ratio of vascular wall thickness to external diameter [WA%, (43.9 +/- 5.3)%] and pulmonary artery media thickness [PAMT, (10.0 +/- 0.7) microm] were significantly higher in H(7) group than those in C group [(16.6 +/- 1.6) mm Hg, (36.3 +/- 4.8)% and (8.5 +/- 1.3) microm respectively, q value were 5.591, 4.082, 2.929, respectively, all P < 0.05]. These parameters reached a high level and remained stable on H(14) group, and RVHI was significantly higher in H(14) group [(27.6 +/- 1.4)%] than in C group [(23.6 +/- 2.9)%, q = 5.817, P < 0.05]. HIF-1alpha protein was barely positive in C group (0.080 +/- 0.009), but markedly up-regulated in H(3) group (0.196 +/- 0.018, compared with C group q = 18.864, P < 0.05), reaching its peak in H(7) group (0.203 +/- 0.022), and then declined slightly in H(14) and H(21) group. HIF-1alpha mRNA increased marginally in H(14) group (0.176 +/- 0.019, compared with C group q = 5.401, P < 0.05, 0.139 +/- 0.017). PHD1 and PHD2 mRNA (0.260 +/- 0.031, 0.196 +/- 0.023) and protein (0.244 +/- 0.030, 0.205 +/- 0.025) were positive in C group. PHD2 mRNA and protein were up-regulated in H(3) group (0.246 +/- 0.023, 0.235 +/- 0.025, compared with C group q value was 5.268, 3.046, respectively, all P < 0.05), reaching its peak in H(14) group whereas PHD1 protein declined in H(14) group (0.210 +/- 0.023, compared with C group q = 3.885, P < 0.05) without significant mRNA change. PHD3 mRNA and protein were detected at low level in C group (0.110 +/- 0.013, 0.153 +/- 0.019), but markedly up-regulated in H(3) group (0.259 +/- 0.024, compared with C group q = 15.831, P < 0.05), and then PHD3 mRNA remained at high level while PHD3 protein declined in H(14) and H(21) group (0.206 +/- 0.025, 0.189 +/- 0.019, compared with H(7) group q value was 6.441, 8.526, respectively, all P < 0.05). Linear correlation analysis showed that HIF-1alpha mRNA and protein were positively correlated with mPAP. There was a positive correlation between HIF-1alpha protein and PHD2, PHD3 mRNA (r value was 0.580, 0.690, respectively, all P value was 0.000) but a negative correlation between HIF-1alpha protein and PHD2 protein (r = -0.704, P < 0.05). CONCLUSIONS: HIF-1alpha was regulated mainly at the protein level during the development of hypoxic pulmonary hypertension. PHD2 and PHD3 are inducible by hypoxia, possibly via elevated HIF-1alpha, suggesting that a hypoxic up-regulation of PHD acts via feedback mechanism to attenuate hypoxia induced responses. PHD may also be regulated by posttranscriptional mechanisms.

Differential and reciprocal regulation between hypoxia-inducible factor-alpha subunits and their prolyl hydroxylases in pulmonary arteries of rat with hypoxia-induced hypertension.

Hypoxia-inducible factor (HIF)-alpha subunits (HIF-1alpha, HIF-2alpha and HIF-3alpha), which play a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH), are regulated through post-translational hydroxylation by their three prolyl hydroxylase domain-containing proteins (PHD1, PHD2 and PHD3). PHDs could also be regulated by HIF. But differential and reciprocal regulation between HIF-alpha and PHDs during the development of HPH remains unclear. To investigate this problem, a rat HPH model was established. Mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. HIF-1alpha and HIF-2alpha mRNA increased slightly after 7 d of hypoxia, but HIF-3alpha increased significantly after 3 d of hypoxia. The protein expression levels of all three HIF-alpha were markedly upregulated after exposure to hypoxia. PHD2 mRNA and protein expression levels were upregulated after 3 d of hypoxia; PHD1 protein declined after 14 d of hypoxia without significant mRNA changes. PHD3 mRNA and protein were markedly upregulated after 3 d of hypoxia, then the mRNA remained at a high level, but the protein declined after 14 d of hypoxia. In hypoxic animals, HIF-1alpha proteins negatively correlated with PHD2 proteins, whereas HIF-2alpha and HIF-3alpha proteins showed negative correlations with PHD3 and PHD1 proteins, respectively. All three HIF-alpha proteins were positively correlated with PHD2 and PHD3 mRNA. In the present study, HIF-alpha subunits and PHDs showed differential and reciprocal regulation, and this might play a key pathogenesis role in hypoxia-induced pulmonary hypertension.