Acute respiratory distress syndrome (ARDS) is a multifactorial syndrome that leads to increased morbidity and mortality in infants and children. The identification of novel biomarkers is critical for the treatment of ARDS. The present study aimed to investigate the effects of chitinase-3-like-1 protein (CHI3L1 or YKL-40) in an in vitro model of ARDS and to explore the potential underlying mechanisms. The in vitro model of ARDS was established in A549 alveolar epithelial type II cells, which were treated by lipopolysaccharide (LPS) to induce inflammation. Transfection was performed to alter YKL-40 expression. The mRNA and protein expression of YKL-40 was determined using reverse transcription-quantitative PCR and western blotting, respectively. Cell Counting Kit-8 and TUNEL assays were used to evaluate the cell viability and apoptosis, respectively. The production of cytokines was evaluated using specific ELISA kits. The relationship between YKL-40 and Fos-related antigen 1 (Fra-1) was verified using luciferase reporter and chromatin immunoprecipitation assays. The expression of the apoptotic proteins was detected using western blotting. The expression levels of YKL-40 and Fra-1 were increased in LPS-treated A549 cells. Higher levels of pro-inflammatory cytokines and induction of cell apoptosis were observed in LPS-treated A549 cells compared with the control. YKL-40 knockdown in LPS-treated A549 cells significantly decreased the production of pro-inflammatory cytokines and reduced cell apoptosis, whereas it concomitantly caused upregulation of Bax and downregulation of Bcl-2, cleaved caspase-3 and cleaved caspase-9. In addition, Fra-1 could directly bind to YKL-40 promoter and regulate its expression level. Overexpression of YKL-40 partly decreased the inhibitory effects of Fra-1 knockdown on the inflammatory response and induction of apoptosis. In summary, the findings from the present study indicated that Fra-1 could bind to YKL-40 and regulate its expression, whereas YKL-40 knockdown could further suppress LPS-induced inflammatory response and apoptosis in A549 cells. These data may provide novel evidence on the diagnosis and therapy of ARDS.

Aim This study aimed to explore the link between neutrophil extracellular traps (NETs) and childhood asthma, to investigate the ability of nitric oxide (NO) to induce NETs in asthmatic children and find inhibitors to reduce NETs in the NO synthesis pathway. Methods A total of 49 children with mild persistent asthma were included in the study and 20 healthy children's blood samples were collected as healthy controls. Children with asthma were divided into symptomatic and asymptomatic groups according to the presence or absence of symptoms on the day of blood collection. Neutrophils in peripheral blood were isolated and plasma was preserved. NO donor (sodium nitroferricyanide(III) dehydrate [SNP]) could provide NO and proved by a fluorescent probe. A PicoGreen Kit was used to detect the NETs quantificationally. Fluorescence microscopy prepared to observe the main structures of NETs. We measured NETs components (extracellular free double-stranded DNA [dsDNA]) in healthy, symptomatic and asymptomatic groups' plasma samples, and we compared the ability of SNP with phosphate-buffered saline, lipopolysaccharides (LPS), and phorbol 12-myristate 13-acetate (PMA) to induce NETs. NO synthase (NOS) inhibitors were added to see the impact on NETs formation. Results Plasma was obtained from all blood samples of 69 children. The neutrophils of 40 asthmatic and 20 healthy children were successfully obtained, the recovery rate was over 95%, and the cell activity was over 80%. There was higher extracellular free dsDNA in the plasma of symptomatic group (n = 27) than asymptomatic group (n = 22) and healthy group (n = 20; P < .05). Studies on neutrophils from 40 children with asthma found that NO can be produced by adding SNP, PMA, and LPS. SNP could induce NETs with dose- and time-dependent. PMA (160 nM) had the strongest ability to induce NETs, LPS (200 ng/mL) followed, SNP (200 mu M) was the weakest (P < .05), and the amount of NETs in the asthma group was significantly higher than that in the healthy group (P < .05). NOS inhibitors had the same blocking capacity for PMA- and LPS-induced NETs (P > .05), while NG-nitro-l-arginine methyl ester (500 mu M) had the strongest inhibitory effect on SNP induction with time-dependent (P < .05). Inducible NOS was found in the NETs structure. Conclusion Children with asthma had higher levels of NETs in peripheral blood, especially when they had asthma symptoms. We verified the ability of NO to induce NETs, and found neutrophils from asthmatic children can produce more NETs in vitro. NOS inhibitors blocked this process may provide new therapeutic targets for childhood asthma.

Neuroinflammation plays a key role in the occurrence and development of neurodegenerative diseases. Microglia, the resident immune cells in the brain, have been recognized to contribute to neuroinflammation. Previous studies have shown that activated mast cells may be involved in surgery-induced neuroinflammation and neuronal apoptosis by using pharmacological methods. This study is aimed at ascertaining the exactly role of mast cells on neuroinflammation with the mast cell-deficient mice. Adult male C57BL6/J wild-type (WT) and mast cell-deficient (C57BL6/J KitWsh/Wsh (Wsh)) mice underwent tibial fracture surgery. Blood-brain barrier (BBB) breakdown, microglial activation, and neuroinflammatory levels were examined at 1 day after surgery. Surgery-induced BBB breakdown, microglial activation, and neuroinflammatory levels were significantly, pharmacologically reduced using a mast cell stabilizer, cromolyn sodium in WT mice (P < 0.05). These results were reproduced with mast cell deficiency. WT mice administered intraventricularly with cromolyn exhibited reduced BBB breakdown, microglial activation, and neuroinflammatory levels versus vehicle (P < 0.05). But there was no effect of cromolyn versus vehicle in Wsh mice, clarifying the specificity of cromolyn on brain mast cells. These findings demonstrated that activated mast cells promote surgery-induced BBB breakdown and neuroinflammation in mice, and open up a new therapeutic target for neuroinflammation-related diseases.

BACKGROUND:The shRNA lentiviral vector was constructed to silence c-Ski expression in cardiac mus- cle cells, with the aim of exploring the role of c-Ski in transforming growth factor b1 (TGF-b1)-induced epithelial-mesenchymal transitions (EMT) in H9C2 cells.METHODS:Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect c-Ski ex- pression at protein and messenger ribonucleic acid (mRNA) levels in 5 different cell lines. Then, lentiviral vector was constructed to silence or overexpress c-Ski in H9C2 cells. MTT and/or soft agar assay and tran- swell assay were used to detect cell proliferation and migration, respectively. The expression levels of c-Ski under different concentrations of TGF-b1 stimulation were detected by RT-qPCR and immunocytochemi- cal analysis. In the presence or absence of TGF-b1 stimulation, the proteins' expression levels of a-SMA, FN and E-cadherin, which are closely correlated with the process of EMT, were measured by western blot after c-Ski silencing or overexpression. Meanwhile, the effect of c-Ski on Samd3 phosphorylation with TGF-b1 stimulation was investigated.RESULTS:There is a high expression of c-Ski at protein and mRNA levels in H9C2 cell line, which first demonstrated the presence of c-Ski expression in H9C2 cells. Overexpression of c-Ski significantly increased H9C2 cell proliferation. The ability of c-Ski gene silencing to suppress cell proliferation was gradually enhanced, and inhibition efficiency was the highest after 6 to 7 d of transfection. Moreover, H9C2 cells with c-Ski knockdown gained significantly aggressive invasive potential when compared with the control group. TGF-b1 stimulation could dose-independently reduce c-Ski expression in H9C2 cells and lead to obvious down-regulated expression of E-cadherin. Interestingly, c-Ski could restore E-cadherin expression while suppressing a-SMA and/or FN expression stimulated by TGF-b1. How- ever, shRNA-induced c-Ski knockdown aggravated only the TGF-b1-induced EMT. Moreover, c-Ski- -shRNA also promoted the phosphorylation of Samd3 induced by TGF-b1.CONCLUSIONS:c-Ski expression in cardiac muscle cells could be down-regulated by TGF-b1. Silencing of c-Ski gene was accompanied by down-regulation of E-cadherin, up-regulation of a-SMA and/or FN and Smad3 phosphorylation induced by TGF-b1, promoting EMT process. Therefore, c-Ski may be closely associated with TGF-b1-induced EMT and play an important role in cardiac fibrosis develop- ment and progression.

Background: Contralateral seventh cervical nerve transfer (contralateral C7 transfer) is a novel treatment for patients with spastic paralysis, including stroke and traumatic brain injury. However, little is known on changes in plasticity that occur in the intact hemisphere after C7 transfer. An appropriate surgical model is required. New method: We described in detail the anatomy of the C7 in a mouse model. We designed a pretracheal route by excising the contralateral C6 lamina ventralis, and the largest nerve defect necessary for direct neurorrhaphy was compared with defect lengths in a prespinal route. To test feasibility, we performed in-vivo surgery and assessed nerve regeneration by immunofluorescence, histology, electrophysiology, and behavioral examinations. Results: Two types of branching were found in the anterior and posterior divisions of C7, both of which were significantly larger than the sural nerve. The length of the nerve defect was drastically reduced after contralateral C6 lamina ventralis excision. Direct tension-free neurorrhaphy was achieved in 66.7% of mice. The expression of neurofilament in the distal segment of the regenerated C7 increased. Histological examination revealed remyelination. Behavioral tests and electrophysiology tests showed functional recovery in a traumatic brain injury mouse. Comparison with existing methods: This is the first direct tension-free neurorrhaphy mouse model of contralateral C7 transfer which shortened the time of nerve regeneration; previous models have used nerve grafting. Conclusions: This paper describes a simple, reproducible, and effective mouse model of contralateral C7 transfer for studying brain plasticity and exploring potential new therapies after unilateral cerebral injury.

Diabetic nephropathy has been proved to be correlated with the occurrence of cardiovascular diseases in diabetic patients. However, the mechanism is unclear. LncRNA Gm4419 has been reported to participate in the development of diabetic nephropathy, while its involvement in cardiac diseases is still unknown. Therefore, our study aimed to investigate the correlation between Gm4419 expression and incidence of coronary heart disease (CHD) and stroke. A total of 100 type 2 diabetic patients combined with diabetic nephropathy and 100 type 2 diabetic patients without diabetic nephropathy were included and followed up for 5years. Renal function and cardiac function indicators as well as serum levels of GM4419 were measured on the day of admission and at the end of follow-up. Occurrence of CHD and stroke was recorded during follow-up and compared between two groups. Renal function of type 2 diabetic patients combined with diabetic nephropathy was worse than that of type 2 diabetic patients without diabetic nephropathy, while no significant differences in cardiac function indicators were found between two groups. Incidences of CHD and stroke were significantly higher in type 2 diabetic patients with diabetic nephropathy than in patients without nephropathy. High serum level of Gm4419 was closely correlated with the occurrence of CHD and stroke as well as poor renal and cardiac functions. LncRNA Gm4419 can promote the development of cardiac diseases in type 2 diabetic patients with diabetic nephropathy.

CCR7 is a G protein-coupled chemokine receptor. In this study, we used immunohistochemistry with tissue microarrays to measure CCR7 expression in tumor specimens from 122 patients with gastric cancer. We show that CCR7 expression is associated with lymph node metastasis (P = 0.022) and overall survival (OS; P = 0.025), and is an independent factor associated with poorer overall survival (P = 0.032). The CCR7 mechanism was predicted based on bioinformatic analysis and verified in gastric cancer cell lines and primary tumor samples. The data show that CCR7 contributes to TGF-β1-induced epithelial-mesenchymal transition (EMT) and that the effects of TGF-β1 are inhibited by a CCR7 neutralizing antibody or a NF-κB inhibitor. Increased TGF-β1 expression was accompanied by nuclear localization of NF-κB-p65 and higher levels of the mesenchymal marker vimentin in human gastric cancer samples. We conclude that the CCR7 axis mediates TGF-β1-induced EMT via crosstalk with NF-κB signaling, facilitating lymph node metastasis and poorer overall survival in patients with gastric cancer. These findings suggest CCR7 is a novel prognostic indicator and a potential target for gastric cancer therapy.

Immunosenescence contributes to pathogenesis of Alzheimer's disease (AD) in the elderly. In this study, we explored the effects of young wild type (WT) splenocytes (ySCs) on Alzheimer's disease by transplanting ySCs into APPswe/PSENldE9 transgenic mice. Young WT splenocytes not only prevented AD, but also improved the spatial learning and memory of APPswe/PSENldE9 transgenic mice. Young WT splenocytes enhanced Aβ clearance, decreased astrogliosis and increased systemic growth differentiation factor 11 (GDF11) levels. Splenocytes derived from old AD mouse promoted AD. There was an increased number of regulatory T cells (Tregs) among old AD splenocytes. We suggest that alterations of GDF11 and Tregs are involved in AD progression and that rejuvenation of the immune system is a potential therapeutic strategy in AD.

Anti-amyloid-β (Aβ) immunotherapy is a potential therapeutic strategy to reduce amyloid plaques and amyloid-associated pathologies in Alzheimer's disease (AD). Immune senescence with aging has also played a crucial role in AD pathogenesis and influences the effect of anti-Aβ immunotherapy. In this study, a combined treatment of Aβ₁₋₄₂-bone marrow-derived dendritic cells (BMDCs) with intraperitoneal injection of splenocytes from young mice was designed as a novel immunotherapy for AD in APPswe/PSEN1de9 transgenic mice models. The results showed that the combined treatment not only elevated the level of anti-Aβ antibodies but also reduced amyloid plaques in brain and finally ameliorated deterioration of spatial learning and memory in AD mice. Additionally, the results revealed an increase of CD68 positive microglial cells in the vicinity of amyloid plaques in the mouse brain, which was responsible for the enhanced phagocytosis of Aβ plaques. In conclusion, the Aβ₁₋₄₂-BMDCs plus splenocytes treatment improved the phagocytosis of microglia and prevented AD pathology more effectively. This combined immunotherapy provided a promising treatment in preventing the progression of AD in clinical studies in the near future.

Ghrelin, a newly discovered bioactive peptide, initially was identified as a strong stimulant for the release of growth hormone (GH) and that has improved cardiac function in patients suffering from end-stage chronic heart failure. Increasing evidence has demonstrated that ghrelin may have myocardial protective effects. However, the role of ghrelin in the pathogenesis of cardiovascular diseases remains unclear. In this study, an in vivo model of rat myocardial calcification induced by vitamin D3 and nicotine was used to study the possible mechanism in the regulatory action of ghrelin on the calcified myocardium. Calcification increased total Ca2+ content and 45Ca2+ deposition in the myocardium and alkaline phosphatase (ALP) activation in the plasma. Compared with the control group, ghrelin mRNA expression was up-regulated and the myocardium calcium content was significantly increased in vitamin D3 and nicotine-treated rats. Rats were subcutaneously injected with 1 or 10 nmol/kg ghrelin. Rats treated with both low- and high-dose ghrelin decreased total Ca2+ content and 45Ca2+ deposition in cardiac muscle and inhibited ALP activation in the myocardium and plasma, in a concentration-dependent manner. In addition, osteopontin (OPN) mRNA expression significantly decreased and that of endothelin (ET-1) significantly increased with myocardial calcification. Ghrelin treatment increased OPN expression at the mRNA level and reduced ET-1 mRNA expression in a dose-dependent manner. These results indicate that exogenous administration with ghrelin attenuates myocardial calcification induced by nicotine and vitamin D3, and that the possible mechanism is via the ghrelin-induced increase in the OPN mRNA levels and decrease in the ET-1 mRNA expression in the myocardium.