Identification of Hub Genes in the Pathogenesis of Bronchiolitis Obliterans via Bioinformatic Analysis and Experimental Verification.

Background: Bronchiolitis obliterans (BO) is a chronic disease that can arise as a complication of severe childhood pneumonia and can also impact the long-term survival of patients after lung transplantation. However, the precise molecular mechanism underlying BO remains unclear. We aimed to identify BO-associated hub genes and their molecular mechanisms. Methods: BO-associated transcriptome datasets (GSE52761, GSE137169, and GSE94557) were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). Additional bioinformatics analyses, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI) analyses, were performed to determine functional roles and DEG-associated regulatory networks. Prediction of hub genes using the 12 algorithms available in the Cytohubba plugin of Cytoscape software was also performed. Verification was performed using the BO mouse model. Results: Our results revealed 57 DEGs associated with BO, of which 18 were down-regulated and 39 were up-regulated. The Cytohubba plugin data further narrowed down the 57 DEGs into 9 prominent hub genes (CCR2, CD1D, GM2A, TFEC, MPEG1, CTSS, GPNMB, BIRC2, and CTSZ). Genes such as CCR2, TFEC, MPEG1, CTSS, and CTSZ were dysregulated in 2,3-butanedione-induced BO mice, whereas TFEC, CTSS, and CTSZ were dysregulated in nitric acid-induced BO mouse models. Conclusion: Our study identified and validated four novel BO biomarkers, which may allow further investigation into the development of distinct BO diagnostic markers and novel therapeutic avenues.

A Chemiluminescence Enzyme Immunoassay Based on Biotinylated Nanobody and Streptavidin Amplification for Diazinon Sensitive Quantification.

The advantages of genetic modification and preferable physicochemical qualities make nanobody (Nb) easy to develop a sensitive and stable immunosensor platform. Herein, an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) based on biotinylated Nb was established for the quantification of diazinon (DAZ). The anti-DAZ Nb, named Nb-EQ1, with good sensitivity and specificity, was obtained from an immunized library via a phage display technique, where the molecular docking results indicated that the hydrogen bond and hydrophobic interactions between DAZ and complementarity-determining region 3 and framework region 2 in Nb-EQ1 played a critical role in the Nb-DAZ affinity processes. Subsequently, the Nb-EQ1 was further biotinylated to generate a bi-functional Nb-biotin, and then an ic-CLEIA was developed for DAZ determination via signal amplification of the biotin-streptavidin platform. The results showed that the proposed method based on Nb-biotin had a high specificity and sensitivity to DAZ, with a relative broader linear range of 0.12-25.96 ng/mL. After being 2-folds dilution of the vegetable samples matrix, the average recoveries were 85.7-113.9% with a coefficient of variation of 4.2-19.2%. Moreover, the results for the analysis of real samples by the developed ic-CLEIA correlated well with that obtained by reference method GC-MS (R2 ≥ 0.97). In summary, the ic-CLEIA based on biotinylated Nb-EQ1 and streptavidin recognition demonstrated itself to be a convenient tool for the quantification of DAZ in vegetables.

A juvenile murine model with chronic lung inflammation induced by repeated intratracheal instillation of lipopolysaccharides: a versatile and replicable model.

Background: Recurrent lower respiratory tract infection or chronic pulmonary infection often occur in children with chronic lung diseases (CLDs). By continuous lung inflammation, recurrent and chronic infection could cause irreversible airway structural and lung function damage, which eventually leads to respiratory failure and death. Methods: In purpose of recapitulating persistent high-intensity lung inflammation caused by recurrent lower respiratory tract infection or chronic infection, we established a juvenile murine model with chronic lung inflammation induced by repeated intratracheal instillations of lipopolysaccharides (LPS) from Pseudomonas aeruginosa once a week for 4 weeks. Four-week-old C57BL/6N mice were divided into 4 groups, including LPS0.5 group (n=15), LPS1.0 group (n=15), Control group (n=15) and Normal group (n=15). Mice in LPS0.5 group and LPS1.0 group were instilled intratracheally with 0.5 mg/kg LPS and 1.0 mg/kg LPS respectively. Mice in control group were instilled intratracheally with LPS-free sterile 0.9% NaCl, whereas normal group received no treatment. The successful chronic lung inflammation murine model was validated via (I) pathological manifestations of chronic inflammatory mononuclear-cell infiltration and lung parenchyma damage; (II) decreased lung function. Results: All mice in LPS1.0 group died before the third instillation. No death after instillation was observed in Control and LPS0.5 group. Histological analysis revealed that in LPS0.5 group, 7 days after the third instillation, most bronchus and parabronchial vessels were wrapped by infiltrating monocytes and lymphocyte and alveolar cavities were compressed, which were not observed in control and normal group. Also, ratio of forced expiratory volume in 0.1 second (FEV0.1) and forced vital capacity (FVC) in LPS0.5 group was significantly lower (P<0.0001) than both control group and normal group, suggesting ventilatory dysfunction developed after repeatedly intratracheal instillation once a week for 4 weeks. Conclusions: Intratracheal instillation of 0.5 mg/kg LPS once a week for 4 weeks can cause chronic lung inflammation in young mice.

Ascorbic Acid Derivative 2-O-ß-d-Glucopyranosyl-l-Ascorbic Acid from the Fruit of Lycium barbarum Modulates Microbiota in the Small Intestine and Colon and Exerts an Immunomodulatory Effect on Cyclophosphamide-Treated BALB/c Mice.

2-O-ß-d-Glucopyranosyl-l-ascorbic acid (AA-2ßG) is a natural and stable ascorbic acid derivative isolated from the fruits of Lycium barbarum. In our present study, cyclophosphamide (Cy) was used to make BALB/c mice immunosuppressive and AA-2ßG was used to intervene immunosuppressive mice. It was found that Cy treatment resulted in a series of changes on basic immune indexes including a decrease of thymus and spleen indexes and levels of pro-inflammatory cytokines and destruction of leucocyte proportion balance, accompanied with weight loss, reduction in colon length, and changes of hepatic function markers. However, all these changes were reversed in varying degrees by AA-2ßG intervention. Notably, AA-2ßG could significantly change both mouse colonic and small-intestinal microbiota. The key responsive taxa found in a mouse colon were Muribaculaceae, Ruminococcaceae, Oscillibacter, Rikenella, Helicobacter, Negativibacillus, Alistipes, and Roseburia, and the key responsive taxa found in a mouse small intestine were Muribaculaceae, Anaerotruncus, and Paenibacillus. The results demonstrated that AA-2ßG could modulate microbiota in the small intestine and colon and exert an immunomodulatory effect. Further studies should focus on the degradation pathways of AA-2ßG and the interaction between AA-2ßG and Muribaculaceae.

Long-Term Consumption of 2-O-ß-d-Glucopyranosyl-l-ascorbic Acid from the Fruits of Lycium barbarum Modulates Gut Microbiota in C57BL/6 Mice.

The modulating effect of 2-O-ß-d-glucopyranosyl-l-ascorbic acid (AA-2ßG), a natural derivative of ascorbic acid from the fruits of Lycium barbarum, on mice gut microbiota was investigated in the present study. It was found that AA-2ßG was able to adjust the structure of mice gut microbiota, elevated the relative abundances of Verrucomicrobia, Porphyromonadaceae, Verrucomicrobiaceae, and Erysipelotrichaceae, and meanwhile reduced the relative abundances of Firmicutes, Lachnospiraceae, Rikenellaceae, Ruminococcaceae, Bdellovibrionaceae, Anaeroplasmataceae, and Peptococcaceae. Through the linear discriminant analysis effect size analysis, the key microbiota that were found to be significantly changed after long-term consumption of AA-2ßG were Ruminococcaceae, Porphyromonadaceae, Lachnospiraceae, and Rikenellaceae. In addition, AA-2ßG could upregulate pro-inflammatory cytokines, promote tight junctions between intestinal cells, facilitate the generation of short-chain fatty acids (SCFAs), and upregulate the mRNA expression level of SCFAs receptors, indicating that AA-2ßG might promote organism health. The results demonstrated that AA-2ßG might maintain organism health by modulating gut microbiota.

Effects of long-term intake of anthocyanins from Lycium ruthenicum Murray on the organism health and gut microbiota in vivo.

The relationship between diet, especially polyphenols, and health has been receiving increasing attention. Polyphenols were degraded by gut microbiota into metabolites and acted on the body to exert many bioactivities from several targets such as antioxidative stress, anti-inflammation, intestinal barrier and gut microbiota modulation. After long-term treatment of mice with anthocyanins from Lycium ruthenicum Murray (ACN), antioxidant status in liver (T-AOC, T-SOD, CAT, GSH and GSH-Px were increased and AST, ALT, ALP and MDA were decreased), anti-inflammatory status in colon (the expression of mRNA of iNos, Cox-2, Tnf-α, Il-6, Il-1ß and Ifn-γ were significantly reduced), intestinal barrier (the expression of mRNA of Zo-1, Occludin, Claudin-1 and Muc1 were significantly increased) and gut microbiota (Barnesiella, Alistipes, Eisenbergiella, Coprobacter and Odoribacter were proliferated) were all regulated in ACN group. Meanwhile, the content of short-chain fatty acids in cecal contents and feces were increased. Taken together, long-term intake of ACN could promote organism healthy and these results have important implications for the development of ACN as a functional food ingredient.

Modulation of gut microbiota by Ilex kudingcha improves dextran sulfate sodium-induced colitis.

Increasing evidence shows that the gut microbiota contributes to the occurrence and development of colitis. Kudingcha (KDC), made from the leaves of Ilex kudingcha, could mitigate inflammation, however, little is known about the relationship between modulatory effect on gut microbiota by KDC and improvement of colitis. In this study, the attenuating effects of KDC extract (KDCE) on dextran sulfate sodium (DSS)-induced colitis and gut microbiota in C57BL/6 mice were investigated. It was found that the supplementation of KDCE could alleviate typical symptoms of IBD including weight loss, colon shortening, intestinal barrier damage, and decreases in the colitis disease activity index and pro-inflammatory cytokines. Moreover, KDCE supplementation could reverse the alteration of gut microbiota in the colitic mice by increasing the abundances of potential beneficial bacteria, e.g. Odoribacter, Prevotella and Helicobacter, and decreasing the abundances of potential harmful bacteria, e.g. Parabacteroides, Bacteroides, Turicibacter, Parasutterella and Lachnospiraceae. The levels of short-chain fatty acids in feces, cecum contents and serum were also regulated by KDCE. Furthermore, the correlation analysis suggested that KDCE could attenuate DSS-induced colitis which might be related to the alteration of gut microbiota. Therefore, the modulation of gut microbiota by KDCE might be a potential strategy for improving inflammation-driven diseases.

2-O-ß-d-Glucopyranosyl-l-ascorbic Acid, an Ascorbic Acid Derivative Isolated from the Fruits of Lycium Barbarum L., Modulates Gut Microbiota and Palliates Colitis in Dextran Sodium Sulfate-Induced Colitis in Mice.

In this study, the effects of 2-O-ß-d-glucopyranosyl-l-ascorbic acid (AA-2ßG), a natural ascorbic acid derivative from the fruits of Lycium barbarum, on treating the dextran sulfate sodium (DSS)-induced colitis in mice were investigated. The results revealed that AA-2ßG had palliating effects on DSS-induced inflammatory bowel disease (IBD) in terms of slowing down the trends of body weight and solid fecal mass loss, reducing colitis disease activity index, improving serum physiological and biochemical indicators, increasing colon length, blocking proinflammatory cytokines, and increasing tight junction proteins. Additionally, AA-2ßG treatment could promote the production of short-chain fatty acids and modulate the composition of the gut microbiota. The key bacteria related to IBD were found to be Porphyromonadaceae, Prevotellaceae, Rikenellaceae, Parasutterella, Parabacteroides, and Clostridium. The results indicated that AA-2ßG might treat IBD through the regulation of gut microbiota, suggesting that AA-2ßG has the potential to be used as a dietary supplement in the treatment of IBD.