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Bronchial asthma poses a considerable burden on both individual patients and public health. Sesamin is a natural lignan that relieves asthma. However, the potential regulatory mechanism has not been fully validated. In this study, we revealed the mechanism of sesamin in inhibiting airway inflammation of asthma. In cockroach extract (CRE)-induced asthmatic mice, sesamin efficiently inhibited inflammatory cell infiltration, expressions of total and CRE-specific IgE in serum, and inflammatory cytokines (including IL-4, 5, 13) in bronchoalveolar lavage fluid. Further study revealed that sesamin inhibited Th2 cells in the mediastinal lymph nodes and spleen, the expression of PTEN-induced putative kinase 1 (PINK1) and Parkin, and apoptosis of lung airway epithelial cells.

The aim of this study is to investigate the protective effects of glaucocalyxin A (GLA) on airways in mouse models of asthma, concerning the inflammatory mediators, Th1/Th2 subgroup imbalance, and Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. Hematoxylin and eosin/periodic acid–Schiff staining was used to observe the pathological changes in lung tissues. Inflammatory cytokine contents in the bronchoalveolar lavage fluid were detected by enzyme-linked immunosorbent assay. Protein expression levels were detected with Western blot, immunohistochemistry, and immunofluorescence. In vivo studies showed that, in ovalbumin (OVA)-induced asthmatic mouse models, the GLA treatments reduced the airway hyperresponsiveness and the secretion of inflammatory cells, declined the proliferation of goblet cells, decreased the levels of IL-4, IL-5, and IL-13, and increased the contents of interferon-γ and IL-12. Moreover, GLA inhibited the protein expression levels of TLR4, MyD88, TRAF6, and NF-κB in OVA-induced asthmatic mouse models. Further in vitro studies showed that GLA inhibited the expression of NF-κB, p-IκBα, tumor necrosis factor-α, IL-6, and IL-1β and blocked the nuclear transfer of NF-κB in lipopolysaccharide-stimulated RAW264.7 macrophages. Conclusively, GLA can inhibit the inflammatory responses in OVA-induced asthmatic mice and inhibit the release of inflammatory factors in LPS-induced RAW264.7 macrophages, which may be related to the inhibition of TLR4/NF-κB signaling pathway.

Background Asthma is characterized by chronic inflammation and airway remodeling. However, limited study is conducted on the gene expression profiles of ovalbumin (OVA) induced asthma in mice. Here, we explored the gene expression profiles in lung tissues from mice with OVA-induced asthma using microarray and bioinformatics analysis. Methods For establishment of OVA-induced asthma model, mice first received intraperitoneal sensitization with OVA on day 0, 7 and 14, followed by atomizing inhalation of OVA 3 times a week for 8 weeks. The lung tissues were collected and subjected to microarray analysis, bioinformatics analysis and expression validation. Results Microarray data of lung tissues suggested that 3754 lncRNAs and 2976 mRNAs were differentially expressed in lung tissues between control and asthmatic mice, including 1647 up-regulated and 2106 down-regulated lncRNAs, and 1201 up-regulated and 1766 down-regulated mRNAs. GO analysis displayed that the up-regulated genes were enriched in inflammatory response, leukocyte migration involved in inflammatory response, and Notch signaling pathway. KEGG pathway analysis indicated that the enriched pathway terms of the up-regulated gene included Toll-like receptor signaling pathway and Th17 cell differentiation signaling pathway. Additionally, based on the previously published literatures on asthma and inflammation, we screened out down-regulated genes, such as Smg7, Sumo2, and Stat5a, and up-regulated genes, such as Myl9, Fos and Tlr4. According to the mRNA-lncRNA co-expression network, we selected lncRNAs associated with above genes, including the down-regulated lncRNAs of NONMMUT032848, NONMMUT008873, NONMMUT009478, and NONMMUT006807, and the up-regulated lncRNAs of NONMMUT052633, NONMMUT05340 and NONMMUT042325. The expression changes of the above genes were validated in lung tissues by real-time quantitaive PCR and Western blot. Conclusions Overall, we performed gene microarray on lung samples from OVA-induced asthmatic mice and summarized core mRNAs and their related lncRNAs. This study may provide evidence for further research on the therapeutic targets of asthma.

Introduction Pterostilbene (Pts) may be used for allergic asthma treatment. The AMPK/Sirt1 and Nrf2/HO‐1 pathways are potential targets for asthma treatement. However, the relationship between Pts and AMPK/Sirt1 and Nrf2/HO‐1 pathways in asthma is unclear. Herein, we aim to explore the pharmacological effects of Pts on oxidative stress and allergic inflammatory response as well as the mechanism involving AMPK/Sirt1 and Nrf2/HO‐1 pathways. Methods Asthma model was established in mice with ovalbumin (OVA). The model mice were treated by different concentrations of Pts. Lung pathological changes were observed through histological staining. In vitro, lipopolysaccharide (LPS)‐stimulated 16HBE cells were treated with Pts. The siAMPKα2, siSirt1 and siNrf2 knockdown, and treatment with compound C, EX‐527 or ML385 were also performed in 16HBE cells. Enzyme‐linked immunosorbent assay was used to detect interleukin‐4 (IL‐4), IL‐13, IL‐5, total and OVA specific immunoglobulin E (IgE), and interferon γ (IFN‐γ). Pneumonography was used to measure the airway hyperreactivity (AHR). Superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels were also detected. Immunohistochemistry, Western blot and immunofluorescence were used to measure protein levels. Results Pts significantly attenuated lung inflammatory cell infiltration and goblet cell proliferation. Meanwhile, Pts treatment could reduce IL‐4, IL‐13, IL‐5, and IgE (total and OVA specific) levels in the asthma model mice. However, IFN‐γ in bronchoalveolar lavage fluid was elevated. In addition, Pts reduced AHR. We also found that Pts treatment promoted serum SOD and CAT, and reduced MDA. In vitro results showed that Pts treatment promoted iNOS, TNF‐α, COX‐2, IL‐1β, and IL‐6 expressions in 16HBE cells, prolonged G0/G1 phase of the cell cycle, and resulted in a shortened G2M phase. Moreover, we found that Pts promoted the phosphorylation of AMPK in 16HBE, and meanwhile inhibited the increase of ROS induced by LPS. Additionally, Pts treatment inhibited p‐AMPK, Sirt1, Nrf2 and HO‐1, which in turn leads to the alleviation of AMPK/Sirt1 and Nrf2/HO‐1 pathways. Conclusion Pts alleviated oxidative stress and allergic airway inflammation via regulation of AMPK/Sirt1and Nrf2/HO‐1 signaling pathways., Pts can relieve asthma by suppressing oxidative stress through the AMPK/Sirt1 and Nrf2/HO‐1 signaling pathways. Thus, Pts might be a potential anti‐inflammatory drug for the treatment of asthma.

Brain diseases including brain tumor, Alzheimer's disease, Parkinson's disease, etc. are difficult to treat. The blood-brain barrier (BBB) is a major obstacle for drug delivery into the brain. Although nano-package and receptor-mediated delivery of nanomedicine markedly increases BBB penetration, it yet did not extensively improve clinical cure rate. Recently, brain extracellular space (ECS) and interstitial fluid (ISF) drainage in ECS have been found to determine whether a drug dissolved in ISF can reach its target cells. Notably, an increase in tortuosity of ECS associated with slower ISF drainage induced by the accumulated harmful substances, such as: amyloid-beta (Aβ), α-synuclein, and metabolic wastes, causes drug delivery failure.The methods of nano-package and receptor-mediated drug delivery and the penetration efficacy of nanomedicines across BBB and ECS are assessed.Invasive delivering drug via ECS and noninvasive near-infrared photo-sensitive nanomedicines may provide a promising benefit to patients with brain disease.

Bronchial asthma is characterized by chronic airway inflammation, airway hyperresponsiveness, and airway remodeling. MicroRNA (miRNA) has recently been implicated in the pathogenesis of asthma. However, the mechanisms of different miRNAs in asthma are complicated, and the mechanism of miRNA-182-5p in asthma is still unclear. Here, we aim to explore the mechanism of miRNA182-5p in asthma-related airway inflammation. Ovalbumin (OVA)-induced asthma model was established. MiRNA Microarray Analysis was performed to analyze the differentially expressed miRNAs in the asthma model. We found that the expression of miRNA-182-5p was significantly decreased in OVA-induced asthma. In vitro, IL-13 stimulation of BEAS-2B cells resulted in a significant up-regulation of NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4), accompanied by mitochondrial damage-induced apoptosis, NLRP3 (NOD-like receptor family pyrin domain-containing 3)/IL-1β activation, and reduced miRNA-182-5p. In contrast, overexpression of miRNA-182-5p significantly inhibited epithelial cell apoptosis and NLRP3/IL-1β activation. In addition, we found that miRNA-182-5p could bind to the 3’ untranscripted region of NOX4 mRNA and inhibit epithelial cell inflammation by reducing oxidative stress and mitochondrial damage. In vivo, miRNA-182-5p agomir treatment significantly reduced the percentage of eosinophils in bronchoalveolar lavage fluid, and down-regulated Th2 inflammatory factors, including IL-4, IL-5, and OVA induced IL-13. Meanwhile, miRNA-182-5p agomir reduced the peribronchial inflammatory cell infiltration, goblet cell proliferation and collagen deposition. In summary, targeting miRNA-182-5p may provide a new strategy for the treatment of asthma.

This study is to investigate the inhibitory effects and mechanisms of DEK‐targeting aptamer (DTA‐64) on epithelial mesenchymaltransition (EMT)‐mediated airway remodelling in mice and human bronchial epithelial cell line BEAS‐2B. In the ovalbumin (OVA)‐induced asthmatic mice, DTA‐64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA‐64 reduced collagen deposition, transforming growth factor 1 (TGF‐β1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α‐SMA), as well as weekend matrix metalloproteinases (MMP‐2 and MMP‐9) and NF‐κB p65 activity. In the in vitro experiments, we used TGF‐β1 to induce EMT in the human epithelial cell line BEAS‐2B. DEK overexpression (ovDEK) or silencing (shDEK) up‐regulated or down‐regulated TGF‐β1 expression, respectively, on the contrary, TGF‐β1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF‐β1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF‐β1‐mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA‐64 against EMT of asthmatic mice and BEAS‐2B might partially be achieved through suppressing TGF‐β1/Smad, MAPK and PI3K signalling pathways. DTA‐64 may be a new therapeutic option for the management of airway remodelling in asthma patients.

Background: Macrophages substantially shape the development, progression, and complications of inflammation-driven diseases. Although numerous researches support a critical role for Notch signaling in most inflammatory diseases, there is limited data on the role of Notch signaling in TLR4-induced macrophage activation and the interaction of Notch signaling with other signaling pathways(e.g., the NF-kB pathway during inflammation) in macrophages. Methods and Results: In this study, we confirmed that stimulation with TLR4 ligand LPS up-regulates Notch1 expression in RAW264.7 monocyte/macrophage-like cell line. LPS also induced the expression of Notch target genes Notch1 and Hes1 mRNA in macrophages, suggesting that TLR4 signaling enhances Notch pathway activation. The upregulation of Notch1, NICD, and Hes1 protein by LPS treatment was inhibited by the Notch1 inhibitor of DAPT. The increase of TNF-a, IL-6, and IL-1b induced by LPS was inhibited by DAPT while jagged1, the Notch1 ligand, rescued them. Furthermore, the suppression of Notch signaling by DAPT up-regulated CYLD expression but down-regulated TRAF6, IKKa/bphosphorylation, and subsequently phosphorylation and degradation of IκB-α, indicating the inhibition of TLR4-triggered NF-kB activation by DAPT. Interestingly, DAPT showed no inhibitory effect on the increase of MyD88 expression induced by LPS in our study. Conclusions: Our study shows that the stimulation of macrophages via the TLR4 signaling cascade triggers the activation of Notch1 signaling, which regulates the expression patterns of genes involved in pro-inflammatory responses through by activating NF-kB. It may be dependent on the CYLD-TRAF6-IKK pathway. The Notch1 signaling may be considered as a potential therapeutic target against infectious and inflammatory driven diseases.

We investigated the regulatory role of miR-181b-5p in neutrophilic asthma and its mechanisms by targeting DEK. DEK, matrix metalloproteinase (MMP)-2, and MMP-9 were overexpressed and the miR-181b-5p was decreased in mice with neutrophilic asthma. DEK was a direct target of miR-181b-5p. In mouse model, miR-181b-5p agomir had an inhibitory effect on airway inflammation and remodeling. miR-181b-5p inhibited DEK/p-GSK-3β

MicroRNAs (miRNAs) can participate in airway remodeling by regulating immune molecule expression. Here, we aimed to identify the differential miRNAs involved in airway remodeling. Airway remodeling was induced by ovalbumin in female BALB/C mice. The differentially expressed miRNAs were screened with microarray. GO (Gene Ontology) and KEGG enrichment analysis was performed. The miRNA target gene network and miRNA target pathway network were constructed. Verification with real-time PCR and Western blot was performed. We identified 63 differentially expressed miRNAs (50 up-regulated and 13 down-regulated) in the lungs of ovalbumin-induced airway remodeling mice. Real-time PCR confirmed that 3 miRNAs (mmu-miR-1931, mmu-miR-712-5p, and mmu-miR-770-5p) were significantly up-regulated, and 4 miRNAs (mmu-miR-128-3p, mmu-miR-182-5p, mmu-miR-130b-3p, and mmu-miR-20b-5p) were significantly down-regulated. The miRNA target gene network analysis identified key mRNAs in the airway remodeling, such as Tnrc6b (trinucleotide repeat containing adaptor 6B), Sesn3 (sestrin 3), Baz2a(bromodomain adjacent to zinc finger domain 2a), and Cux1 (cut like homeobox 1). The miRNA target pathway network showed that the signal pathways such as MAPK (mitogen-activated protein kinase), PI3K/Akt (phosphoinositide 3-Kinase/protein kinase B), p53 (protein 53), and mTOR (mammalian target of rapamycin) were closely related to airway remodeling in asthma. Collectively, differential miRNAs involved in airway remodeling (such as mmu-miR-1931, mmu-miR-712-5p, mmu-miR-770-5p, mmu-miR-128-3p mmu-miR-182-5p, and mmu-miR-130b-3p) as well as their target genes (such as Tnrc6b, Sesn3, Baz2a, and Cux1) and pathways (such as MAPK, PI3K/Akt, p53, mTOR pathways) have been identified. Our findings may help to further understand the pathogenesis of airway remodeling.

Neutrophils, classified as professional phagocytes, are crucial in killing bacteria and preventing inflammation. When studying the roles of neutrophils in the development of the septic peritonitis induced by E. coli, we noticed some of the larger cells existed among peritoneal lavage fluid cells (PLCs). Besides the large size, their nuclei are segmented and flat, and squeezed to the marginal zone of the inner membrane. The cells, therefore, were designated as E. coli induced larger neutrophils (e-Neus). Further studies showed that, the e-Neus were ly6G positive, indicating the e-Neus were a type of neutrophils. The enlarged cell size and marginal nucleus of the e-Neus were caused by engulfing abundant of E. coli, marking the active participation of the e-Neus in clearance of E. coli. Functionally, the e-Neus generated reactive oxygen species (ROS) and IL-10. Furthermore, the occurrence and accumulation of the e-Neus were closely correlated with the severity of septic peritonitis and mortality of the mice. Overall, the e-Neus presented here may enrich the understandings on neutrophil transitions in response to various insults, and could be used to evaluate the severity of septic peritonitis induced by E. coli.

We investigated whether Panax notoginseng saponin (PNS-R1) attenuates allergic rhinitis (AR) through AMPK/Drp1-mediated mitochondrial fission. AR model was established in mice by Ovalbumin (OVA). In vitro, human nasal epithelial cells (HNEpCs) were stimulated using recombinant human interleukin 13 (IL-13). PNS-R1 was administrated in vivo and in vitro. Then, HE staining of nasal tissue, ELISA detection of immunoglobulin E (IgE) and proinflammatory cytokine levels in serum and nasal lavage fluid, flow cytometry analysis of Th1/Th2 ratio and apoptosis, TUNEL staining, Western blot, detection of reactive oxygen species (ROS) and mitochondrial ROS, immunofluorescence analysis of Tom20 and mitochondrial fission protein Drp1 co-localization, and mitochondrial membrane potential detection, were performed. PNS-R1 attenuated allergic symptoms in AR mice, decreased OVA-specific IgE, IL-4, IL-6, IL-8, IL-13, and TNF-α levels, and restored the Th1/Th2 imbalance. Meanwhile, we found that PNS-R1 treatment significantly reduced apoptosis, ROS production, and co-localization of Tom20 and Drp1 in the nasal epithelium of AR mice. In vitro, we found that PNS-R1 upregulated mitochondrial membrane potential and reduced ROS and mitochondrial ROS production as well as Cleaved-caspase-3/9, Bax, Cyt-c, Apaf-1 expression and mitochondrial fission. Mechanistically, we found that PNS-R1 downregulated Drp1 phosphorylation (Ser 616) and Drp1 translocation in an AMPK-dependent manner, promoted MFN2 expression, and reduced TXNIP, NLRP3, Caspase-1, and IL-1β expression. PNS-R1 may protect mitochondrial integrity by inhibiting AMPK/Drp1 and TXNIP/NLRP3 signaling pathway, thereby alleviating AR symptoms in mice. PNS-R1 may have great potential as a therapeutic agent for AR.

This study is to investigate the role of Sphingosine-1-phosphate (S1P) in the asthma progression, and the involvement of autophagy. Airway remodeling mice were subjected to the HE, PAS, and Masson staining. Protein expression levels in the tissues, samples and model cells were detected with ELISA, Western blot analysis, and immunohistochemical/immunofluorescent analysis. The S1P2 receptor antagonist JTE-013 decreased the inflammatory cell infiltration and goblet cell production in asthmatic mice tissues. The IL-1, IL-4, IL-5 and serum IgE contents were decreased in bronchoalveolar lavage fluid, while the Beclin1 expression in lung tissues was decreased. The LC3B1 to LC-3B2 conversion was decreased, with increased P62 accumulation and decreased p-P62 expression. In airway remodeling mice, JTE-013 significantly decreased collagen deposition in lung tissues and decreased smooth muscle cell smooth muscle activating protein expression. In lung tissue, the expression levels of Beclin1 were decreased, with decreased LC3B1 to LC-3B2 conversion, as well as the increased P62 accumulation and decreased p-P62 expression. However, these effects were reversed by the RAC1 inhibitor EHT 1864. Similar results were observed for the silencing of S1P2 receptor in the cells, as shown by the decreased Beclin1 expression, decreased LC3B1 to LC-3B2 conversion, increased P62 accumulation, and decreased p-P62 expression. The smooth muscle activators were significantly decreased in the JTE-013 and EHT1864 groups, and the EHT 1864 + S1P2-SiRNA expression level was increased. S1P is involved in the progression of asthma and airway remodeling, which may be related to the activation of S1PR2 receptor and inhibition of autophagy through RAC1.

Lymphocyte activation gene-3 (LAG3) is a transmembrane protein expressed on activated T cells and delivers inhibitory signals to render the T cells unable to effectively help B cells to produce antibodies to microbes and vaccines. Presumably, antagonizing LAG3 could enhance the antibody responses to vaccines, and LAG3 antagonists could facilitate vaccines to induce vigorous antibody responses. In this study, we designed a LAG3-interfering antisense oligonucleotide, designated as LIO-1. The LIO-1 is complementary to an identical region shared in human and mouse LAG3 mRNA. We demonstrated that LIO-1 induced the degradation of LAG3 mRNA in immune cells, decreased the LAG3 expression on CD4+ T cells, maintained the prolonged proliferation and promoted the activation of antigen-specific CD4+ T cells, and increased the production of IFN-γ, IL-2, and IL-6 in the antigen re-stimulated immune cells. In addition, we found that LIO-1 enhanced the antibody responses induced by ISA35-formulated recombinant antigen vaccine or ISA35-formulated inactivated influenza virus vaccines in mice. Thus, the LIO-1, a nucleic acid LAG3 antagonist, could facilitate vaccines to induce vigorous antibody responses and has the possibility to be used as a novel adjuvant.

// Li Hua 1 , Mingli Fang 2 , Boqi Dong 1 , Sheng Guo 2 , Cuiyun Cui 1 , Jiwei Liu 2 , Yun Yao 2 , Yue Xiao 2 , Xin Li 2 , Yunjia Ren 2 , Xiuping Meng 1 , Xu Hao 1 , Peiyan Zhao 2 , Yilan Song 1 , Liying Wang 2 , Yongli Yu 1 1 Department of Immunology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, Jilin 130021, China 2 Department of Molecular Biology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, Jilin 130021, China Correspondence to: Liying Wang, email: wlying@jlu.edu.cn Yongli Yu, email: ylyu@jlu.edu.cn Keywords: NKG2DL, allogeneic tumors, early stage, NK cells, NKG2D + cells Received: October 12, 2015 Accepted: June 09, 2016 Published: July 19, 2016 ABSTRACT Allogeneic tumors are eventually rejected by adaptive immune responses, however, little is known about how allogeneic tumors are eradicated at the early stage of tumor development. In present study, we found that NKG2DL low expressing cancer cells were developed into palpable allogeneic tumors in mice within a week after the inoculation, while NKG2DL high expressing cancer cells failed to. The NKG2DL high expressing cancer cells could increase NKG2D + NK cells in the allogeneic mice after being inoculated for 3 days. Artificially up-regulating NKG2DL on cancer cells with low level expressed NKG2DL by a CpG ODN resulted in the retardation and rejection of the allogeneic tumors at the early stage. The contribution of up-regulated NKG2DL to the early rejection was further confirmed by the results that the development of allogeneic tumors from cancer cells transfected with NKG2DL genes was significantly inhibited in mice at the early stage. Overall, hopefully, the data may provide insights for combining the allogeneic NK cell adoptive transfer with the approaches of up-regulating NKG2DL to treat cancer patients.