Systemic Responses of BALB/c Mice to <i>Salmonella typhimurium</i> Infection

<i>Salmonella typhimurium</i> is a bacterial pathogen that poses a great threat to humans and animals. In order to discover hosts’ responses to <i>S. typhimurium</i> infection, we collected and analyzed biofluids and organ tissues from mice which had ingested <i>S. typhimurium</i>. We employed ¹H NMR spectroscopy coupled with multivariate data analysis and immunological techniques. The results indicate that infection leads to a severe impact on mice spleen and ileum, which are characterized by splenomegaly and edematous villi, respectively. We found that increased levels of itaconic acid were correlated with the presence of splenomegaly during infection and may play an important role in <i>Salmonella</i>-containing vacuole acidification. In addition, metabonomic analyses of urine displayed the development of salmonellosis in mice, which is characterized by dynamic changes in energy metabolism. Furthermore, we found that the presence of <i>S. typhimurium</i> activated an anti-oxidative response in infected mice. We also observed changes in the gut microbial co-metabolites (hippurate, TMAO, TMA, methylamine). This investigation sheds much needed light on the host–pathogen interactions of <i>S. typhimurium</i>, providing further information to deepen our understanding of the long co-evolution process between hosts and infective bacteria

Gut Microbiota Composition Modifies Fecal Metabolic Profiles in Mice

The gut microbiome is known to be extensively involved in human health and disease. In order to reveal the metabolic relationship between host and microbiome, we monitored recovery of the gut microbiota composition and fecal profiles of mice after gentamicin and/or ceftriaxone treatments. This was performed by employing ¹H nuclear magnetic resonance (NMR)-based metabonomics and denaturing gradient gel electrophoresis (DGGE) fingerprint of gut microbiota. The common features of fecal metabolites postantibiotic treatment include decreased levels of short chain fatty acids (SCFAs), amino acids and primary bile acids and increased oligosaccharides, d-pinitol, choline and secondary bile acids (deoxycholic acid). This suggests suppressed bacterial fermentation, protein degradation and enhanced gut microbial modification of bile acids. <i>Barnesiella</i>, <i>Prevotella</i>, and <i>Alistipes</i> levels were shown to decrease as a result of the antibiotic treatment, whereas levels of <i>Bacteroides</i>, <i>Enterococcus</i> and <i>Erysipelotrichaceae incertae sedis</i>, and <i>Mycoplasma</i> increased after gentamicin and ceftriaxone treatment. In addition, there was a strong correlation between fecal profiles and levels of <i>Bacteroides</i>, <i>Barnesiella</i>, <i>Alistipes</i> and <i>Prevotella</i>. The integration of metabonomics and gut microbiota profiling provides important information on the changes of gut microbiota and their impact on fecal profiles during the recovery after antibiotic treatment. The correlation between gut microbiota and fecal metabolites provides important information on the function of bacteria, which in turn could be important in optimizing therapeutic strategies, and developing potential microbiota-based disease preventions and therapeutic interventions

Metabolomics Insights into the Modulatory Effects of Long-Term Low Calorie Intake in Mice

There is increasing evidence that calorie restriction without malnutrition can extend longevity and delay the onset of age-associated disorders. Identifying the biochemical perturbations associated with different dietary habits would provide valuable insights into associations between metabolism and longevity. To reveal the effects of long-term dietary interventions on metabolic perturbations, we investigated serum and urinary metabolic changes induced by interactive high/low fat diet in combination with/without reduced caloric intake over a life span in mice using NMR-based metabonomics. We found that the high calorie dietary regime disturbed lipid metabolism, suppressed glycolysis and TCA cycles, stimulated oxidative stress, promoted nucleotide metabolism and gluconeogenesis, and perturbed gut microbiota–host interactions. Such changes could be modified by long-term low calorie intake. Most importantly, we found that the calorie intake index exerts a dominant effect on metabolic perturbations irrespective of dietary regime. Our investigation provides a holistic view of the metabolic impact of long-term dietary interventions, which are important for detecting physiological changes and dietary effects on mammalian metabolism

Streptozotocin-Induced Dynamic Metabonomic Changes in Rat Biofluids

Diabetes mellitus is a complex polygenic disease caused by gene-environment interactions with multiple complications, and metabonomic analysis is crucial for pathogenesis, early diagnosis, and timely interventions. Here, we comprehensively analyzed the dynamic metabolic changes in rat urine and plasma, which were induced by the well-known diabetogenic chemical streptozotocin (STZ), using ¹H NMR spectroscopy in conjunction with multivariate data analysis. The results showed that a single intraperitoneal injection of STZ with a moderate dosage (55 mg/kg) induced significant urinary metabonomic changes within 24 h. These changes showed time-dependence and heterogeneity among the treated animals with an animal recovered within 11 days. STZ-induced metabonomic alterations were related to suppression of glycolysis and TCA cycle, promotion of gluconeogenesis and oxidation of amino acids, alterations in metabolisms of basic amino acids associated with diabetic complications, and disruption of lipid metabolism and gut microbiota functions. With diffusion-edited NMR spectral data, we further observed the STZ-induced significant elevation of monounsaturated fatty acids and total unsaturated fatty acids together with reductions in PUFA-to-MUFA ratio in the blood plasma. These findings provided details of the time-dependent metabonomic changes in the progressive development of the STZ-induced diabetes mellitus and showed the possibility of detecting the biochemical changes in the early stage of type 1 diabetic genesis

E2F1/CDK1 expression is necessary for trophic deprivation-induced neuronal apoptosis.

<p>Rat cortical neurons were co-transfected with expression plasmids for ß-galactosidase with, either empty vector or vector expressing E2F1 or CDK1 (A and B). Similarly, ß-galactosidase plasmid was co-transfected along with scrambled, E2F1 or CDK1 shRNAs (C, D and E) and the extent of apoptosis was examined 48 h after transfection, or after an additional 24 h of trophic deprivation (TD) induction post 48 h transfection. <b>A</b>. Neurons transfected with E2F1 vector (0.6 µg DNA/0.5×10⁶ neurons) increased basal apoptosis as compared to empty vector. <b>B</b>. Neurons transfected with CDK1 (0.8 µg DNA/0.5×10⁶ neurons) vector enhanced TD-induced apoptosis as compared to empty vector transfected cells. <b>C–D</b>. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons transfected with two different shRNAs targeting either E2F1 or CDK1. <b>E</b>. Attenuation of TD-induced chromatin condensation in neurons transfected by CDK1 shRNA1 is shown. Representative photomicrographs of the shRNA transfected neurons, which also co-express GFP protein from the separate promoter are shown. In all experiments ß-galactosidase was co-transfected at 0.01 µg DNA/0.5×10⁶ neurons to visualize transfected neurons at later apoptotic stage. Neurons with either normal diffuse chromatin morphology or apoptotic condensation of nuclei (after Hoechst 33258 chromatin staining) are indicated by arrowheads or arrows, respectively. Statistical analysis was performed by Kruskal-Wallis one-way ANOVA on ranks, followed by post hoc adjustments using Dunnett's test. For A ^*p<0.001, vs. vector; For C ^*p<0.01, vs. TD vector; For D ^*p<0.05, vs. TD vector; Error bars are ± S.E.M. from three independent experiments.</p

CR8 administration reduces SCI-induced neuronal death.

<p><b>A</b>. CR8 attenuated caspase 3 and fodrin cleavage, the latter as indicated by reduction of the 145/150 kDa fragment immunoreactivity. <b>B–C</b>. Signal quantification using densitometry and normalization relative to GAPDH levels demonstrated that the observed changes are significant. The figures reflect representative western blots. N = 4. ^*p<0.05 vs. sham. <b>D–E</b>. TUNEL analysis was performed on 20 µm sections from rats at 24 hours post-SCI using an ApopTag® Fluorescein/Red detection kit. TUNEL/NeuN staining revealed that CR8 effectively reduced neuronal death in the epicenter of the injury (p<0.05), as well as in rostral and caudal sections located in two consecutive 2 mm distance regions from the epicenter. Representative images in panel D were shown at 2 mm caudal to epicenter. Scale bar = 100 µm. <b>F</b>. Six to eight representative animals - were selected from each treatment group for quantification of neurons (NeuN⁺ cells). Unbiased stereology by optical fractionators method using StereoInvestigator Software (MBF Biosciences) indicates that CR8 administration significantly increased number of surviving neurons within 10 mm zone surrounding the injury epicenter (p<0.05, vs vehicle).</p

Pharmacological inhibition of CDK1 blocks neuronal apoptosis.

<p>Cortical neurons were pre-treated with Roscovitine or CR8 (CDK1 inhibitors) or vehicle and then exposed to TD-or campthotecin induced apoptosis. <b>A</b>. Representative photomicrographs of control and trophic deprived neurons treated with the indicated concentrations Roscovitine and CR8 are shown. Upper row presents phase contrast images (Healthy neurons are indicated by larger cell bodies and abundant processes; Apoptotic neurons display shrunken cell bodies and sparse or lost processes). Lower row shows chromatin staining with Hoechst 33258. Arrows and arrowheads indicate surviving and apoptotic neurons, respectively suggesting an attenuation of TD-induced neuronal death in neurons pre-treated with Roscovinine or CR8. <b>B</b>. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons pre-treated with Roscovitine (10 µM; ^*p<0.05, vs. TD vehicle) whereas CR8 at concentrations as low as 1 µM (^***p<0.001, vs. TD vehicle) almost completely blocked development of apoptotic features in neuronal nuclei. <b>C</b>. Significant attenuation of campthotecin-induced apoptosis in neurons pre-treated with Roscovitine (50 µM; ^*p<0.001, vs. vehicle) and CR8 at concentrations as low as 1 µM (^***p<0.001, vs. vehicle).</p

SCI-induced immunoreactivity of E2F1 and CDK1 was attenuated by CR8 treatment.

<p><b>A</b>. Coronal section in intact spinal cord (a–c) shows that E2F1 was expressed in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of E2F1 (d–f) was increased, and highly expressed by motor neurons. The upregulation of E2F1 was clearly attenuated by CR8 treatment (g–i). <b>B</b>. In intact spinal cord (a–c), CDK1/NeuN was detected in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of CDK1 (d–f) was increased, and highly expressed by motor neurons. CDK1 upregulation was attenuated by CR8 treatment (g–i). All images were taken at 2 mm rostral to epicenter. Scale bar = 100 µm for C–F.</p

The temporal profile and cell specificity of E2F1 and CDK1 expression after SCI.

<p><b>A–B</b>. Coronal section in intact spinal cord (A) showed that E2F1 is relatively weak and detected mainly in neurons of the gray matter. At 24 h after SCI, E2F1 immunoreactivity was upregulated not only in gray matter but also in lesion area (B). C. E2F1⁺ cells were also co-labelled with NeuN in the dorsal horn of the gray matter at 1 day after SCI. D–E. Only a small subset of E2F1⁺ cells in the lesion area were positive for OX42 at 24 h (D) and 7 d (E) after SCI. F–G. In the intact spinal cord (F), CDK1 immunoreactivity is relatively weak and detected mainly in motor neurons in the ventral horn and CC1⁺ oligodendrocytes. At 24 h after SCI (G), CDK1 immunoreactivity was upregulated not only in the ventral horn but also in the spared white matter, colocalized with CC1⁺ oligodendrocytes. CDK1⁺ cells also appeared in the lesion area. <b>H–I</b>. CDK1 was expressed by CC1⁺ oligodendrocytes in the white matter in the intact spinal cord (H) and at 1 day after SCI. <b>J</b>. Only a small subset of CDK1⁺ cells in the lesion area were positive for OX42 at 24 h after SCI. <b>K</b>. Coronal section in intact spinal cord (a) shows that E2F1 was expressed in the motor neurons in the ventral horn. Immunoreactivity of E2F1 (b–d) was increased at 5 h, and 1–3 days post injury, and highly expressed by motor neurons. L. In intact spinal cord (a), CDK1/NeuN was detected in the motor neurons in the ventral horn. At 5 h after injury, immunoreactivity of CDK1 (b) was increased and sustained until 3 days post injury (c–d), and highly expressed by motor neurons. All images were taken at 2 mm rostral to epicenter. Scale bar = 500 µm for A–B, F–G. Scale bar = 100 µm for C–E, H–J, K–L.</p

CR8 administration reduces SCI-induced activation of the E2F1/CDK1 signaling pathway.

<p>Samples were obtained from rats exposed to spinal cord injury and CR8 treatment (1 mg/kg intraperitoneal administration) and analyzed by western blotting. Equal protein loading is demonstrated by consistent GAPDH levels. <b>A</b>. CR8 attenuated SCI mediated increase in E2F1 and its target cyclin A expression. <b>B–C</b>. Quantification of respective western blots in panel A. <b>D</b>. CR8 reduced SCI induced increase in phospho-(Ser54)-n-myc, phosphorylated CDK substrates and expression of cyclin B1. <b>E–G</b>. Quantification of respective western blots in panel E. <b>H–J</b>. Administration of CR8 significantly reduced Bim and c-Myb expression at 24 h after SCI. H shows representative Western blots for Bim, c-Myb, and the loading control, GAPDH. I and J show quantitative analysis of Bim and c-Myb expression. N = 4. ^*p<0.05 vs. vehicle group.</p