m6A echoes with DNA methylation: Coordinated DNA methylation and gene expression data analysis identified critical m6A genes associated with asthma.

Asthma is a chronic inflammatory disease that involves complex gene-environment interactions. Methylation of nucleotides, such as 5-methylcytosine (5mC) in DNA and N6-methyladenosine (m6A) in mRNA, carries important information for gene regulation. Our study screened m6A genes and genes associated with asthma from the Gene Expression Omnibus (GEO) databases GSE63383, GSE119580, GSE38003, GSE34313, GSE13168, and GSE35643. GSE52778, GSE35643, GSE40996, and GSE64744), and DNA methylation data from GSE85568 and GSE146377. We screened out 6 m6A related genes (FTO, IGF2BP2, RBM15, RBMX, WTAP, and YTHDC1) that were significantly dysregulated in asthma or proinflammatory conditions. A correlation study showed a high correlation between m6A genes and gene pairs such as WTAP, IL7R, and TLR2; RBMX, SLC22A4, IL33, TNC, FLG, and IL6R (|r| ≥ 0.8). Following DNA methylation dataset analysis, we proposed several DNA methylation-m6A modification asthma-related gene axes such as cg19032951/cg15153914-IGF2BP2-SMAD3. Interestingly, several target genes, such as SMAD3, possess the ability to participate in DNA methylation processes, which may reciprocally regulate the expression of m6A genes and form a closed-loop regulation axis. Some classic DNA methylation-related genes, such as TET1, UHRF1, and ZBTB4, were also involved. We identified an integrated profile of m6A gene expression in asthma and proposed a novel potential interplay between DNA methylation and m6A modification in asthma pathogenesis. Using the CMAP database, we found that resveratrol may target these dysregulated m6A genes, and therefore may serve as a potential therapeutic agent for asthma.

IL-24 deficiency protects mice against bleomycin-induced pulmonary fibrosis by repressing IL-4-induced M2 program in macrophages.

Idiopathic pulmonary fibrosis (IPF) is the most common type of idiopathic interstitial pneumonia and has one of the poorest prognosis. However, the molecular mechanisms underlying IPF progression remain largely unknown. In this study, we determined that IL-24, an IL-20 subfamily cytokine member, was increased both in the serum of IPF patients and the bronchoalveolar lavage fluid (BALF) of mice following bleomycin (BLM)-induced pulmonary fibrosis. As a result, IL-24 deficiency protected mice from BLM-induced lung injury and fibrosis. Specifically, loss of IL-24 significantly attenuated transforming growth factor ß1 (TGF-ß1) production and reduced M2 macrophage infiltration in the lung of BLM-induced mice. Mechanistically, IL-24 alone did not show a perceptible impact on the induction of M2 macrophages, but it synergized with IL-4 to promote M2 program in macrophages. IL-24 suppressed IL-4-induced expression of suppressor of cytokine signaling 1 (SOCS1) and SOCS3, through which it enhanced signal transducer and activator of transcription 6/peroxisome proliferator-activated receptor gamma (STAT6/PPARγ) signaling, thereby promoting IL-4-induced production of M2 macrophages. Collectively, our data support that IL-24 synergizes with IL-4 to promote macrophage M2 program contributing to the development of pulmonary fibrosis.

Assessment of proliferation, migration and differentiation potentials of bone marrow mesenchymal stem cells labeling with silica-coated and amine-modified superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles have been widely used for cell labeling in preclinical and clinical studies, to improve labeling efficiency, particle conjugation and surface modifications are developed, but some modified SPIONs exert side-effect on physiological activity of cells, which cannot be served as ideal cell tracker. In this study, amine-modified silica-coated SPIO (SPIO@SiO2-NH2, SPIO@S-N) nanoparticles were used to label bone marrow derived mesenchymal stem cells (BM-MSCs), then the stem cell potentials were evaluated. It was found BM-MSCs could be efficiently labeled by SPIO@S-N nanoparticles. After labeling, the BM-MSCs viability kept well and the migration ability increased, but the osteogenesis and adipogenesis potentials were not impaired. In steroid associated osteonecrosis (SAON) bone defect model, stem cell implantation was performed by injection of SPIO@S-N labeled BM-MSCs into marrow cavity locally, it was found the SPIO positive cells homed to the periphery of defect region in control group, but were recruited to the defect region in poly lactic-coglycolic acid/tricalcium phosphate (PLGA/TCP) scaffold implantation group. In conclusion, SPIO@S-N nanoparticles promoted migration while retained proliferation and differentiation ability of BM-MSCs, implying this kind of nanoparticles could be served not only an ideal tracking marker but also an accelerator for stem cell homing during tissue repair.

Blockade of JAK2 protects mice against hypoxia-induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation.

OBJECTIVES: Hypoxia is an important risk factor for pulmonary arterial remodelling in pulmonary arterial hypertension (PAH), and the Janus kinase 2 (JAK2) is believed to be involved in this process. In the present report, we aimed to investigate the role of JAK2 in vascular smooth muscle cells during the course of PAH. METHODS: Smooth muscle cell (SMC)-specific Jak2 deficient mice and their littermate controls were subjected to normobaric normoxic or hypoxic (10% O2 ) challenges for 28 days to monitor the development of PAH, respectively. To further elucidate the potential mechanisms whereby JAK2 influences pulmonary vascular remodelling, a selective JAK2 inhibitor was applied to pre-treat human pulmonary arterial smooth muscle cells (HPASMCs) for 1 hour followed by 24-hour hypoxic exposure. RESULTS: Mice with hypoxia-induced PAH were characterized by the altered JAK2/STAT3 activity in pulmonary artery smooth muscle cells. Therefore, induction of Jak2 deficiency in SMCs protected mice from hypoxia-induced increase of right ventricular systolic pressure (RVSP), right ventricular hypertrophy and pulmonary vascular remodelling. Particularly, loss of Jak2 significantly attenuated chronic hypoxia-induced PASMC proliferation in the lungs. Similarly, blockade of JAK2 by its inhibitor, TG-101348, suppressed hypoxia-induced human PASMC proliferation. Upon hypoxia-induced activation, JAK2 phosphorylated signal transducer and activator of transcription 3 (STAT3), which then bound to the CCNA2 promoter to transcribe cyclin A2 expression, thereby promoting PASMC proliferation. CONCLUSIONS: Our studies support that JAK2 could be a culprit contributing to the pulmonary vascular remodelling, and therefore, it could be a viable target for prevention and treatment of PAH in clinical settings.

[The effect of LPS on airway inflammation, airway remodeling and TLR4 expression in asthmatic rat].

OBJECTIVE: To investigate the effects of lipopolysaccharide (LPS) on airway inflammation, airway remodeling and the expression of Toll-like receptor 4 (TLR4) mRNA in asthmatic rats. METHODS: Twenty-four SPF level SD rats were randomly divided into four groups (n = 6): control group, low dose of LPS group, high dose of LPS group and asthma group. Using ovalbumin (OVA) to sensitize and challenge to establish asthmatic rat model. Observed pathological changes of lung tissue by HE staining, inflammatory cell infiltration was observed by airway wall eosinophils (EOS) counts; airway resistance was determined; image analysis software was used to determine the thickness of airway wall, detected airway smooth muscle TLR4 expression levels by RT-PCR. RESULTS: The rat airway resistance and the EOS number of airway wall and the thickness of airway wall in asthma group, low dose of LPS group and high dose of LPS group were significantly higher than those in control group (P < 0.01). The above-mentioned parameters of high dose of LPS group showed significantly lower than those in asthma group and low dose of LPS group (P < 0.05). The expression of rat airway smooth muscle TLR4 mRNA in low dose of LPS group and high dose of LPS group were significantly higher than those in asthma group (P < 0.01). And the expression of rat airway smooth muscle TLR4 mRNA in high dose of LPS group was significantly higher than that in low dose of LPS group (P < 0.05). CONCLUSION: TLR4 plays an important role in asthmatic airway inflammation and airway remodeling, LPS may play double-sided regulation in asthmatic airway inflammation and airway remodeling by activated TLR4.

[Effects of TOLL-like receptor 4 on passively sensitized human airway smooth muscle cells proliferation and synthesis and secretion function of TGF-beta1].

OBJECTIVE: To investigate the activation of Toll like receptor 4 (TLR4) on passively sensitized human airway smooth muscle cells (HASMCs) proliferation and the synthesis and secretion function. METHODS: Through the cultivation of primary HASMCs, we studied TLR4 expression on cell surface, cell proliferation and transformation of parturient factor-beta1 (TGF-beta1) in asthma under the condition of synthesis and secretion level by passively sensitized HASMCs with asthma serum. RESULTS: Compared with the control group, in passive sensitized group and TNF-alpha group TLR4 expression were significantly increased (P < 0.01), significantly enhanced proliferation (P < 0.01), total protein concentration, IgE secretion and TGF-beta1 were significantly higher (P < 0.01); and all the above parameters were increased more significantly in TNF group compared with those in the target effect of passively group; and those parameters were significantly reduced in anti-TLR4 antibody group compared with those in the target effect both of passively sensitized group and TNF-alpha group. CONCLUSION: TLR4 on passively sensitized HASMCs activated can induce the excessive proliferation of HASMCs and a large number of synthesis and secretion of TGF-beta1, resulting in changing airway micro-environment, which involved in airway remodeling in asthma.

[Effect of TLR4 on the migration of asthmatic airway smooth muscle cells induced by airway epithelial cells].

OBJECTIVE: To explore the effect of Toll-like receptor 4 (TLR4) activation on the migration of asthmatic airway smooth muscle cell (ASMCs) induced by airway epithelial cells. METHODS: Primary ASMCs were cultured by the method of cell digestion. Cell culture supernatant of RTE cells were collected by TNF-alpha stimulation of epithelial cells. Detected the IL-8 and RANTES levels in the supernatant. The transmembrane migration of asthmatic ASMCs were detected by Modified Boyden chemotaxis chamber. The effect of TLR4 on the migration of asthmatic ASMCs induced by epithelial cells with TLR4 antibody drugs as a tool. RESULTS: The levels of IL-8 and RANTES in the supernatant of TNF-alpha groups were significantly increased, and that in the 20 ng/ml group was significantly higher than other groups (P < 0.01). The transmembrane migration of asthmatic ASMCs groups was increased than that of control group. The transmembrane migration of asthmatic ASMCs from asthma group and TNF-alpha + TLR4 antibody group was significantly decreased compared with that in TNF-alpha group (P < 0.01). The migration of asthma ASMCs from TNF-alpha + TLR4 antibody group was increased than that of asthma group (P < 0.05). CONCLUSION: TLR4 in the surface of asthmatic ASMCs may be activated by cytokines secreted by the airway epithelial cells and enhance the transmembrane migration of asthmatic ASMCs induced by airway epithelial cells so that it plays a role in airway remodeling of asthma.

[Arsenic trioxide enhances TRAIL inducing human lung cancer cell line A549 cells apoptosis by down-regulate the expression of NF-kappaB].

OBJECTIVE: To study the influence of arsenic trioxide combined with human tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the apoptosis and the expression of NF-kappaB of human non-small-cell lung cancer cell line. METHODS: The proliferation of human non-small-cell lung cancer cell line A549 cultured in vitro were treated by As2O3, TRAIL alone and combined. The cell proliferation was detected by the assay of MTT, flow cytometry with PI stain was used to detect the apoptosis rate, NF-kappaB mRNA level of A549 cells were detected by RT-PCR. The expression of NF-kappaB protein were detected by Western blot. The activity of NF-kappaB was measured by ELISA. RESULTS: Compared with As2O3 alone, As2O3 combined with TRAIL could increase the inhibition and apoptosis ratio significantly (P<0.05). The expression of NF-kappaB in combined group was obviously less than that in As2O3 alone and control; the activity of NF-kappaB was inhibited by combined groups. The NF-kappaB mRNA and protein expression and the activity of NF-kappaB were separately negative related with the apoptosis ratio (P<0.05). CONCLUSION: As2O3 can enhance TRAIL inducing of human lung cancer cell lines A549 apoptosis by inhibition of NF-kappaB signaling pathway.

[Role of toll-like receptor 4 in the asthmatic rat airway smooth muscle cells proliferation and apoptosis].

OBJECTIVE: To explore the role of Toll like receptor 4(TLR4) in the asthmatic rat airway smooth muscle cell (ASMCs) proliferation and apoptosis. METHODS: Established rat model of asthma,isolated and cultured rat ASMCs in asthma, using methods of small molecule RNA interference technology and lipofection method, for small molecule RNA-TLR4 transfection, detected proliferation of ASMCs by MIT minim colorimetry, apoptosis of ASMCs by TUNNEL, the expression of TLR4 protein and mRNA were detected by Western blot and RT-PCR in cells. RESULTS: The proliferation of ASMCs in TNF-alpha group were significantly higher than that in control group and siRNA-TLR4 transfection group and TNF-alpha + siRNA-TLR4 transfection group respectively and the proliferation of ASMCs in siRNA-TLR4 transfction group was lower than that in control group. The apoptosis rate of ASMCs in TNF-alpha group was lower than that in control group, siRNA-TLR4 transfection group and TNF-alpha + siRNA-TLR4 transfection group respectively and the apoptosis rate of ASMCs in siRNA-TLR4 transfection group and TNF-alpha + siRNA-TLR4 transfection group were significantly higher than those in control group. The mRNA and protein expression of TLR4 in control group and TNF-alpha group were significantly higher than those in siRNA-TLR4 transfection group and TNF-alpha + siRNA-TLR4 transfection group. The mRNA and protein expression of TLR4 in TNF-alpha group were significantly higher than those in control group (P < 0.01). CONCLUSION: Activation of TLR4 may contribute to asthmatic airway smooth muscle cell proliferation, inhibiting apoptosis and play an important role in airway remodeling in asthma.