Long-chain non-coding RNA n337374 relieves symptoms of respiratory syncytial virus-induced asthma by inhibiting dendritic cell maturation via the CD86 and the ERK pathway.

BACKGROUND: In this study, we investigated the relationship between long-chain non-coding RNAs (lncRNAs) and respiratory syncytial virus (RSV)-exacerbated asthma. METHODS: Transcriptome microarray was used to detect differentially expressed lncRNAs in dendritic cells (DCs) co-cultured with RSV-infected human airway epithelial cells and DCs infected with RSV. The identified downregulation of lncRNA n337374 was validated using fluorescence RT-qPCR. LncRNA n337374-overexpressing DCs and RSV-exacerbated asthmatic mouse models were established. Airway hyper-reactivity and bronchoalveolar lavage fluid (BALF) were examined, and pathological changes in lung tissues were observed in mice. Surface molecules in DCs were detected by flow cytometry and RT-qPCR and the expression of CD86 and mitogen-activated protein kinases was determined by western blot. RESULTS: In an RSV-exacerbated asthmatic mouse model, the airway wall was thickened, luminal stenosis was observed, a large number of inflammatory cells were infiltrated in the lung tissue, lung function was impaired, and counts of inflammatory cells in the BALF were increased. The overexpression of lncRNA n337374 ameliorated these pathological changes and improved impaired lung function and inflammation in an asthmatic mouse model. In DCs co-cultured with RSV-infected human airway epithelial cells, CD86 expression was promoted and ERK was markedly phosphorylated. When lncRNA n337374-overexpressing DCs were used in the co-cultures, the expression of CD86 and phosphorylated ERK was decreased. CONCLUSION: The results suggest that lncRNA n337374 overexpression may suppress DC maturation by downregulating the CD86 and ERK pathway, subsequently relieving the symptoms of RSV-induced asthma. LncRNA n337374 may be a promising target in the treatment of RSV infection-induced asthma.

Nanoparticle-mediated RNA interference of angiotensinogen decreases blood pressure and improves myocardial remodeling in spontaneously hypertensive rats.

Angiotensinogen (AGT) has been shown to have a role in cardiac hypertrophy, while depletion of the AGT gene in spontaneously hypertensive rats (SHR) has not been investigated. The present study investigated the effect of AGT knockdown on cardiac hypertrophy in SHR. For this, small hairpin (sh)RNAs were intravenously injected into SHRs, using a nanoparticle­mediated transfection system. The experimental rats were divided into the following groups: a) Blank control with water treatment only, b) negative control with biscarbamate­crosslinked Gal­polyethylene glycol polyethylenimine nanoparticles (GPE)/negative shRNA, c) AGT­RNA interference (RNAi) group with GPE/AGT­shRNA, and 4) normotensive control using Wistar­Kyoto rats (WKY) with water treatment. Three and five days following the first injection, the levels of hepatic AGT mRNA and AGT protein as well as plasma levels of AGT were markedly decreased in the AGT­RNAi group (P<0.05). Furthermore, a significant decrease in systolic blood pressure (SBP), left ventricular weight to body weight ratio and heart weight to body weight ratio were observed in the AGT­RNAi group compared with those in the control groups. The depletion of AGT in SHR led to a reduction in SBP by 30±4 mmHg, which was retained for >10 days. Cardiac hypertrophy was also significantly improved in AGT­knockdown rats. In conclusion, the present study showed that AGT­silencing had a significant inhibitory effect on hypertension and hypertensive­induced cardiac hypertrophy in SHRs.

Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative.

Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79-100 nm and zeta potential was 6-15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53-65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery.

Effect of GPE-AGT nanoparticle shRNA transfection system mediated RNAi on early atherosclerotic lesion.

OBJECTIVE: To investigate the effects of RNA interference targeting AGT on early atherosclerotic lesion in the hypertensive state. METHODS: Hypertension and atherosclerosis rats were treated with GPE nanoparticles carrying AGT shRNA. Systolic blood pressure and heart rate were measured for 2 consecutive weeks. Three days after treatment, the mRNA and protein expressions of AGT in the liver were measured by PCR and western blot assay, respectively. The blood levels of AGT and Ang II were determined by ELISA. H&E staining and electron microscopy were performed. RESULTS: Three days after AGT shRNA treatment, the mRNA and protein expressions of AGT in the liver were markedly reduced and the blood levels of AGT and Ang II dramatically decreased as compared to the remaining 3 groups (P < 0.05). Three days after AGT shRNA treatment, the blood pressure was reduced by 27 ± 4 mmHg when compared with that at baseline (P < 0.05). About 11 days after AGT shRNA treatment, the blood pressure began to increase. The blood pressure remained unchanged in the remaining 3 groups. Microscopy showed the atherosclerotic lesions were markedly attenuated in AGT shRNA treated rats but the liver and kidney functions remained stable (P > 0.05) when compared with the remaining 3 groups. CONCLUSION: Transfection with GPE nanoparticle carrying AGT shRNA can stably lower the blood pressure and improve the atherosclerotic lesions which lead to the delayed development of early atherosclerotic lesions in hypertension rats with concomitant atherosclerosis.

Biscarbamate cross-linked polyethylenimine derivative with low molecular weight, low cytotoxicity, and high efficiency for gene delivery.

Polyethylenimine (PEI), especially PEI 25 kDa, has been widely studied for delivery of nucleic acid drugs both in vitro and in vivo. However, it lacks degradable linkages and is too toxic for therapeutic applications. Hence, low-molecular-weight PEI has been explored as an alternative to PEI 25 kDa. To reduce cytotoxicity and increase transfection efficiency, we designed and synthesized a novel small-molecular-weight PEI derivative (PEI-Et, Mn: 1220, Mw: 2895) with ethylene biscarbamate linkages. PEI-Et carried the ability to condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed complete condensation of pDNA at w/w ratios that exceeded three. The particle size of polymer/pDNA complexes was between 130 nm and 180 nm and zeta potential was 5-10 mV, which were appropriate for cell endocytosis. The morphology of PEI-Et/pDNA complexes observed by atomic force microscopy (AFM) was spherically shaped with diameters of 110-190 nm. The transfection efficiency of polymer/pDNA complexes as determined with the luciferase activity assay as well as fluorescence-activated cell-sorting analysis (FACS) was higher than commercially available PEI 25 kDa and Lipofectamine 2000 in various cell lines. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration in three cell lines. Therefore, our results indicated that the PEI-Et would be a promising candidate for safe and efficient gene delivery in gene therapy.

[Peak inspiratory flow generated through different analogue dry powder inhalers in Shenzhen healthy preschool children].

OBJECTIVE: Dry powder inhalers (DPIs) are increasingly being used to deliver drugs for the treatment of asthma. It is known that DPIs require a crucial minimal inspiratory flow. Previous studies have demonstrated that the peak inspiratory flow (PIF, L/min) through a DPI is dependent on the type of device, the age of the patient, and the level of bronchial obstruction. However, the peak inspiratory flow of healthy preschool children in China remains scant in the literature. The present study aimed to analyze the ability of inspiring flow through the resistance state of ordinary use inhaler in Shenzhen healthy preschool children by measuring the peak inspiratory flow through the different analogue dry powder inhalers and go further into the relationship between it and the age, weight and forced expiratory volume of the children. METHOD: A survey in 370 healthy preschool children aged 3 to 6 years (75 children aged 3 years, 104 children aged 4 years, 100 children aged 5 years and 91 children aged 6 years) was carried out in Shenzhen. Peak inspiratory flow (PIF) was measured without and with resistances, which mimicked the internal resistances of several inhalers, Diskus, Turbuhaler, Autohaler, Surehaler by PIF meter (In-check DIAL) and then data PIF-N, PIF-D, PIF-T, PIF-A and PIF-S were obtained. Peak expiratory flow (PEF) was measured by PEF meter (MicroPeak, USA). These two measurements were made in a well-controlled setting, and at least three attempts were recorded to establish maximum achievement. Six spirometry parameters forced vital capacity (FVC), forced expiratory volume at 0.5 second (FEV 0.5), forced expiratory volume at 0.75 second (FEV 0.75), forced expiratory volume at one second (FEV1), maximal mid expiratory flow rate (FEF 25 - 75, PEF were measured by using COSMED spirometry of Italy and the FVC measurements should be around the quality control for spirometry in preschool children which we suggested and published in 2005. All data were expressed as mean +/- SD and analyzed with the statistical software SPSS 12.0 for Windows. Pearson's test was used for calculation of the significances of the correlation coefficients. Variance analysis was used for analysing the variability of inspiratory flows through the inhalers. RESULTS: Results were obtained from 295 children aged 3 - 6 years who successfully finished the tests. The PIF-N, PIF-D, PIF-T, PIF-A and PIF-S were significantly different among the groups aged 3 yrs, 4 yrs, 5 yrs and 6 yrs. The peak inspiratory flow significantly increased with age. The PIF-N, PIF-D, PIF-T, PIF-A and PIF-S in the children of 110 cm height and above were significantly higher than those in the children below 110 cm height, so were the parameters between the children of 120 cm height and above and the children below 120 cm. PIF correlated significantly with age, height and weight and the Pearson coefficient was 0.3 - 0.5. The PIFs in different inhalers varied because of the different inner resistances. The minimum and optimum PIFs in resistances of Diskus, Autohaler and Surehaler could be achieved in almost all subjects, but those in resistances of Turbuhaler could be achieved in only 87.5% subjects, most of whom aged 3 yrs or below 100 cm height. There were good correlations between the PIFs in different resistances and main parameters of ventilation function (FVC, FEV 0.5, FEV 0.75, FEV1, FEF 25 - 75, PEF), PEF was the best among them (Pearson correlative coefficient was 0.6). CONCLUSION: The inspiratory ability of the children can be predicted and assessed by using routine measurement of lung function of normal pre-school children. As to the pre-school children of varying ages, the variety of inspiratory ability should be considered completely in the selection of inhaler used during the treatment. The best inhaler suitable for them should be selected properly in order to obtain the best efficacy of treatment individually.