The effect of Mycoplasma gallisepticum infection on energy metabolism in chicken lungs: Through oxidative stress and inflammation.

Mycoplasma gallisepticum (Mg) causes chronic respiratory disease (CRD) in chickens. However, the effect of Mg infection on energy metabolism in chicken lungs is still unknown. The present study was aimed to investigate the effect of Mg infection on energy metabolism in chicken lungs. Four-weeks-old white leghorn chickens were randomly divided into control group (L1) and Mg infection group (L2). Histopathology, transmission electron microscopy, qRT-PCR and Western blot were used to determine the hallmarks of ultrastructural analysis, inflammation and energy metabolism. Results revealed that Mg infection induced oxidative stress in the chicken lungs and serum cytokine activities were enhanced at the three time points. Chickens infected with Mg revealed abnormal morphology and cellular damage including increased inflammatory cells infiltrate, cellular debris and exudate, mitochondrial and DNA damage in the lungs. The mRNA and protein expression level of inflammation-related genes were significantly increased in L2 group, showing that Mg induced inflammation in chicken lungs. In addition, ATPase activities were reduced in L2 group compared to L1 group. Meanwhile, the expression of energy metabolism related genes were decreased at both mRNA and protein level at all assessed time points, which showed that Mg infection weakened energy metabolism in chicken lungs. In summary, the data suggested that Mg infection induced oxidative stress, inflammation and energy metabolism dysfunction in the chicken lungs, exploring new therapeutic targets and providing a reference for comparative veterinary medicine.

Co-infection of Mycoplasma gallisepticum and Escherichia coli Triggers Inflammatory Injury Involving the IL-17 Signaling Pathway.

Mycoplasma gallisepticum and Escherichia coli are well known respiratory disease-inducing pathogens. Previous studies have reported that co-infection by MG and E.coli causes significant economic loss in the poultry industry. In order to assess the respiratory toxicity of co-infection in chicken lung, we established a co-infection model to investigate changes in the inflammatory cytokines, lung tissue structure, and transcriptome profiles of chicken lung. The results showed that co-infection caused a wider range of immune damage and more severe tissue lesions than single-pathogen infection. Differentially expressed gene (DEG) analysis indicated that 3,115/1,498/1,075 genes were significantly expressed among the three infection groups, respectively. Gene ontology and KEGG analysis showed genes enriched in response to immune response, cytokine-cytokine receptor interaction, and inflammation-related signaling pathways. Among these pathways, IL-17 signaling was found to be significantly enriched only in co-infection. The expression of IL-17C, CIKS, TRAF6, NFκB, C/EBPß, and inflammatory chemokines were significantly up-regulated in response to co-infection. Taken together, we concluded that co-infection increased the expression of inflammatory chemokines in lungs through IL-17 signaling, leading to cilia loss and excessive mucus secretion. These results provide new insights into co-infection and reveal target proteins for drug therapy.

Population pharmacokinetics for danofloxacin in the intestinal contents of healthy and infected chickens.

Avian pathogenic Escherichia coli could cause localized and systemic infection in the poultry, and danofloxacin is usually used to treat avian colibacillosis through oral administration. To promote prudent use of danofloxacin and reduce the emergence of drug-resistant E. coli strains, it is necessary to understand the population pharmacokinetics (PopPK) of danofloxacin in chicken intestines. In this study, reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection was used to detect the concentrations of danofloxacin in the contents of duodenum, jejunum, and ileum of the healthy and infected chickens after single oral administration (5 mg/kg body weight). Then, the PopPK of danofloxacin in intestines were analyzed using NONMEM software. As a result, a two-compartment PK model best described the time-concentration profile of duodenal, jejunal, and ileal contents. Interestingly, absorption rate (Ka ), distribution volume (V), and clearance (CL) for danofloxacin from duodenal, jejunal to ileal contents were sequentially decreased in the healthy chickens. However, the trend of Ka , V, and CL of danofloxacin was changed dramatically in the intestine of infected chickens. Ka and V of danofloxacin in the jejunum were higher than in the ileum and duodenum. Compared with healthy chickens, Ka and V of danofloxacin in the duodenum decreased significantly, while increased in jejunum, respectively. It has been noted that Ka decreased and V increased in the ileum of infected chickens. Besides, CL in the duodenum, jejunum, and ileum of infected chickens was, respectively, lower than those of healthy chickens. Interestingly, the relative bioavailability (F) of danofloxacin in the ileum was relatively higher in both healthy and infected chickens. In addition, F in the duodenal, jejunal, and ileal contents of infected chickens was respectively higher than healthy chickens. In summary, the PopPK for danofloxacin in infected chicken intestines was quite different from healthy chickens. The absorption, distribution, and clearance of danofloxacin in healthy chickens decreased from duodenum to jejunum and to ileum. Moreover, the pharmacokinetic characteristics in the intestine of infected chickens changed significantly, and the pharmacokinetic characteristics in the ileum can be used as a representative of all intestinal segments.

Baicalin ameliorates oxidative stress and apoptosis by restoring mitochondrial dynamics in the spleen of chickens via the opposite modulation of NF-κB and Nrf2/HO-1 signaling pathway during Mycoplasma gallisepticum infection.

Mycoplasma gallisepticum (MG) infection produces a profound inflammatory response in the respiratory tract and evade birds' immune recognition to establish a chronic infection. Previous reports documented that the flavonoid baicalin possess potent anti-inflammatory, and antioxidant activities. However, whether baicalin prevent immune dysfunction is largely unknown. In the present study, the preventive effects of baicalin were determined on oxidative stress generation and apoptosis in the spleen of chickens infected with MG. Histopathological examination showed abnormal morphological changes including cell hyperplasia, lymphocytes depletion, and the red and white pulp of spleen were not clearly visible in the model group. Oxidative stress-related parameters were significantly (P < 0.05) increased in the model group. However, baicalin treatment significantly (P < 0.05) ameliorated oxidative stress and partially alleviated the abnormal morphological changes in the chicken spleen compared to model group. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling assay results, mRNA, and protein expression levels of mitochondrial apoptosis-related genes showed that baicalin significantly attenuated apoptosis. Moreover, baicalin restored the mRNA expression of mitochondrial dynamics-related genes and maintain the balance between mitochondrial inner and outer membranes. Intriguingly, the protective effects of baicalin were associated with the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme oxygenase-1 (HO-1) pathway and suppression of nuclear factor-kappa B (NF-κB) pathway in the spleen of chicken. In summary, these findings indicated that baicalin promoted mitochondrial dynamics imbalance and effectively prevents oxidative stress and apoptosis in the splenocytes of chickens infected with MG.

Baicalin Attenuates Mycoplasma gallisepticum-Induced Inflammation via Inhibition of the TLR2-NF-κB Pathway in Chicken and DF-1 Cells.

BACKGROUND: Previous reports demonstrated that baicalin possesses potential anti-inflammatory properties. The present study was conducted to determine the effects of baicalin against inflammatory responses in chicken and DF-1 cells infected with Mycoplasma gallisepticum (MG). METHODS: An MG infection model was developed in chickens to study the anti-inflammatory mechanism of baicalin. Baicalin was mixed in water at a dose of 450 mg/kg per day, and the treatment is continued for 7 consecutive days. Samples were taken at 1, 4, and 7 days post treatment. RESULTS: By using transmission electron microscopy, ultrastructure of lung and tracheal cells has been examined. It can be seen that the cilia cells in the MG-infected group have pyknosis, degeneration, and necrosis. In the lung tissues, alveolar type-I epithelial cells were severely damaged. In the baicalin-treated group, cilia were swollen, mushroom-shaped edema bubbles formed on the apex, and fused together. Alveolar type I epithelial cells injury was significantly reduced. Compared to MG-infection group, the levels of proinflammatory cytokines IL-1ß and TNF-α were significantly decreased (P < 0.01). The corresponding proteins TLR2 and P-p65 decreased in the baicalin-treated group after 1 (p > 0.05), 4 (p < 0.05), and 7 days (p < 0.05), respectively. CONCLUSION: The results showed that baicalin can interfere with inflammatory injury by suppressing the release of inflammatory cytokines IL-1ß and TNF-α during MG infection both in vivo and in vitro. Meanwhile, baicalin suppressed TLR2-NFκB signaling pathway by inhibiting the phosphorylation of p65 and IκB, thereby affecting the expression of inflammatory factors. The results suggested that baicalin acts as a potential anti-inflammatory agent against MG infection in chicken and DF-1 cells.

Preventive effects of nerve growth factor against colistin-induced autophagy and apoptosis in PC12 cells.

In this study, the preventive effects of NGF against colistin-induced autophagy and apoptosis in PC12 cells have been investigated. Fluorescence microscopy, real-time PCR, transmission electron microscopy (TEM), flow cytometery, and western blotting technique were used. The results showed that large amounts of autophagosomes and apoptotic markers were triggered by colistin. Consistently, a significant increase has been noted at mRNA and protein levels in autophagy and apoptosis-related genes. Besides, TEM analysis showed that autophagic vacuoles were obvious at 12 h, while nuclear chromatin condensation and edge accumulation were clearly seen at 24 h in colistin alone group. Importantly, the visual autophagy and apoptosis were markedly reduced with NGF treatment in a dose-dependent manner. Moreover, colistin-induced reduction in mitochondrial membrane potential was partly attenuated by NGF in a dose dependent manner. In summary, NGF ameliorated colistin-induced apoptosis and autophagy, and partially recovered MMP in PC12 cells.

A Dual Role of P53 in Regulating Colistin-Induced Autophagy in PC-12 Cells.

This study aimed to investigate the mechanism of p53 in regulating colistin-induced autophagy in PC-12 cells. Importantly, cells were treated with 125 µg/ml colistin for 12 and 24 h after transfection with p53 siRNA or recombinant plasmid. The hallmarks of autophagy and apoptosis were examined by real-time PCR and western blot, fluorescence/immunofluorescence microscopy, and electron microscopy. The results showed that silencing of p53 leads to down-regulation of Atg5 and beclin1 for 12 h while up-regulation at 24 h and up-regulation of p62 noted. The ratio of LC3-II/I and autophagic vacuoles were significantly increased at 24 h, but autophagy flux was blocked. The cleavage of caspase3 and PARP (poly ADP-ribose polymerase) were enhanced, while PC-12-sip53 cells exposed to 3-MA showed down-regulation of apoptosis. By contrast, the expression of autophagy-related genes and protein reduced in p53 overexpressing cells following a time dependent manner. Meanwhile, there was an increase in the expression of activated caspase3 and PARP, condensed and fragmented nuclei were evident. Conclusively, the data supported that silencing of p53 promotes impaired autophagy, which acts as a pro-apoptotic induction factor in PC-12 cells treated with colistin for 24 h, and overexpression of p53 inhibits autophagy and accelerates apoptosis. Hence, it has been suggested that p53 could not act as a neuro-protective target in colistin-induced neurotoxicity.

Anti-human fibroblast growth factor-21 monoclonal antibody preparation, characterization and analysis of in vitro bioactivity.

Human fibroblast growth factor 21 (hFGF-21) is involved in numerous metabolic processes and elevated hFGF-21 levels are associated with many metabolic diseases. However, the role hFGF-21 serves in the metabolic system is not fully understood. A humanized anti-hFGF-21 monoclonal antibody (mAb) would provide a novel method for further investigations into the role hFGF-21 serves in the metabolic system and related diseases, which may reveal therapeutic targets for future treatment of these diseases. The present study aimed to prepare an anti-hFGF-21 mAb, followed by identification of its characteristics and bioactivity in vitro. The results of the present study identified that the anti-hFGF-21 mAb (clone 2D8) produced had good specificity, had an immunoglobulin isotype of IgG2b and a titer of 1:1.024×106. hFGF-21 was screened for epitopes using fluorescence-activated cell sorting, which revealed a specific 15 amino acid sequence (YQSEAHGLPLHLPGN) that the anti-hFGF-21 mAb recognized. In vitro bioactivity of anti-hFGF-21 was determined using a glucose uptake assay and by measuring the expression of glucose transporter 1 (GLUT1) messenger RNA (mRNA) in 3T3-L1 adipocytes. This revealed that hFGF-21-dependent glucose uptake and GLUT1 mRNA expression were negatively correlated with increasing levels of the anti-hFGF-21 mAb tested, and that hFGF-21 activity could be overcome by increasing concentrations of the mAb, demonstrating that the mAb has hFGF-21-neutralizing activity in vitro.

[Establishment of bacteria display technology for Fab antibody library screening].

AIM: To establish bacterial display technology for the purpose of Fab antibody library screening, by using six amino acids (CDQSSS) of the amino termimus of NlpA protein to anchore antibodies to the periplasmic side of the bacterial inner membrane. METHODS: The NlpA Leader sequences (encoding CDQSSS) was amplified from pNAD plasmid. The PCR product was subcloned into pComb3 expression vector to generate Fab display vector pBFD. The heavy chains of the Fab gene fragments and the light chains of the anti-human IL-1ß (hIL-1ß) antibody were inserted downstream of the NlpA leader and pelB leader respectively to construct the pBFD-Fab for Fab antibody display. Then pBFD-Fab transformed E.coli DH5α was induced by IPTG to express the Fab antibodies, as detected by flow cytometry (FCM), and positive populations were sorted. Instead of PCR, plasmids were extracted for rescue purpose. The rescue plasmids were retransformed to E.coli DH5α and FCM was performed again. RESULTS: The pBFD-Fab-transformed bacteria were incubated with antigen and antigen specific FITC-antibody, and showed strong fluorescence as detected by FCM in a dose-dependent manner. The rescued pBFD-Fab displayed similar fluorescence intensity, indicating the reliability of this technology. CONCLUSION: The Fab expressed by the bacterial display system folds efficiently and binds to hIL-1ß specifically. The plasmid rescue works well and it can avoid mutation and mis-pairing chains. This bacterial display technology has the stability of antibody expression. This study has used the technology to screen anti-hIL-1ß Fab antibody Library successfully.