Integrative Analysis of Proteomics and Metabolism Reveals the Potential Roles of Arachidonic Acid Metabolism in Hypoxia Response in Mouse Spleen.

High altitude hypoxia stress is the key cause of high-altitude pulmonary edema and spleen contraction. The molecular mechanism of immune response of various tissue systems to hypoxia stress remains lacking. In this study, we applied proteomics combined with metabolomics to explore the key molecular profilings involved in high altitude hypoxia response in the spleen of mice. The results showed that 166 proteins were significantly up-regulated, and only 39 proteins were down-regulated. Bioinformatics analysis showed that mineral absorption, neuroactive ligand-receptor interaction, arachidonic acid metabolism, IL-17 signaling pathway and NOD-like preceptor signaling pathway were significantly enriched in the list of 166 upregulated differentially expressed proteins (DEPs). Among these metabolic pathways, the former three pathways were co-identified in KEGG terms from LC-MS/MS based metabolic analysis. We further found that both arachidonate 15-lipoxygenase and hematopoietic prostaglandin D synthase were upregulated by around 30% and 80% for their protein levels and mRNA levels, respectively. Most downstream metabolites were upregulated accordingly, such as prostaglandin A2 and D2. This study provides important evidence that arachidonic acid metabolism potentially promotes spleen hypoxia response through a combined analysis of proteomics and metabolism, which could bring new insights for the spleen targeted rational design upon arachidonic acid metabolism of new therapies.

Metabolomics study of cat small intestine during the early stage of Toxoplasma gondii oocyst formation identifies potential biomarkers.

Toxoplasma gondii (T. gondii) is a zoonotic intracellular protozoan parasite that can invade, replicate and survive in almost all cells of warm-blooded animals. T. gondii infection threatens the life of the fetus or can cause morbidity in the infant. As the only definitive host of T. gondii, felids spread the pathogen mainly by forming oocysts in the small intestines and discharging the oocysts into the ambient environment, consequently polluting water, vegetables, and meat products. In this study, we used untargeted metabolomics technology to study the changes in metabolites that occurred during the early stage of oocyst formation in the cat small intestine following T. gondii infection and attempted to identify metabolic biomarkers that could potentially be used as diagnostic molecular markers in the future. Domestic cats (Felis catus) were infected with T. gondii Pru tissue cysts, and samples of their small intestinal epithelium were collected at 2 and 4 days post-infection (DPI) for metabolic analysis. LC-MS/MS and multivariate statistical analysis were employed to detect metabolomic signatures that discriminated between the infected and control groups. A total of 1673 ions and 1201 ions were obtained in the positive and negative modes, respectively. Of these ions, 175 were up-regulated and 127 were down-regulated in the positive ion mode; whereas, 123 were up-regulated and 81 were down-regulated in the negative ion mode. Three commonly altered ions (0.74_313.0414 m/z, 8.82_615.2621 m/z and 8.16_325.2362 m/z) were determined to have potential research value. Seventy common metabolic pathways were enriched at two time points, with arginine biosynthesis, pyrimidine metabolism, pantothenate and CoA biosynthesis being the three most significant pathways related to T. gondii. The area under the curve (AUC) of differential metabolites combined with relevant literature analysis showed that N-Methylpelletierine and 3,3-Difluoro-17-methyl-5alpha-androstan-17beta-ol have higher predictability and better potential application value than other metabolites. Our analysis of metabolic markers during the early stage of T. gondii oocyst formation in the small intestine of the definitive host (cat) provided novel insight for understanding oocyst development and a theoretical basis for the application of potential biomarkers.

Staphylococcus aureus induces mammary gland fibrosis through activating the TLR/NF-κB and TLR/AP-1 signaling pathways in mice.

To investigate the TLR-NF-κB/AP-1 pathways in S. aureus infection-induced mammary gland fibrosis, mice were infected with S. aureus isolated from the mammary glands of cows with mastitis. Lactating mice were divided into three groups: control group (CON); PBS control group (PBS) and the S. aureus-treated group (S. aureus). Pathological observations revealed that neutrophil infiltration into mammary gland tissue was obviously induced by S. aureus at the early stage of infection (1-7 d). With persistent S. aureus infection, mammary gland fibrosis developed and was characterized by infiltration and proliferation of macrophage, lymphocyte and fibroblast and ECM hyperplasia (7-21 d). Immunohistochemistry staining showed upregulation of fibrosis associated cytokines viz bFGF and PDGF-BB. Real-time qPCR and Western blot analysis revealed that transcription and translation of TLR2, TLR4, bFGF, PDGF-BB, α-SMA and COL Ⅰ α1 was significantly upregulated by S. aureus. NF-κB p65 and AP-I c-jun were translocated into the nucleus after S. aureus infection. There was no remarkable difference between the CON and PBS groups. The datas indicate that mammary gland fibrosis in mice is induced by S. aureus, which promotes cytokine release and the expression of ECM though activating the TLR/NF-κB p65 and TLR/AP-1 c-jun signaling pathways.

Transcriptome sequencing reveals fibrotic associated-genes involved in bovine mammary fibroblasts with Staphylococcus aureus.

Bovine mammary fibrosis represents a considerable health problem of cows, primarily indicated by lactation failure. Staphylococcus aureus (S. aureus) can cause mammary damage, this multifactorial disease necessitates to identify how and to what extent molecular pathogen defense mechanisms prevent bacterial infections in bovine mammary gland. In this study, we have aimed to determine the transcriptional responses in bovine mammary fibroblasts (BMFBs) induced by S. aureus using bioinformatics analysis to determine whether mRNA expression profile changes between BMFBs activation and quiescence. Established primary BMFBs obtained from healthy Holstein bovine were induced 106 CFU/mL heat-inactivated S. aureus and total RNA was isolated 6 h after treatment. The 574 DEGs were involved in gene ontology (GO) that were immune response, apoptotic process, extracellular region, receptor binding, endopeptidase activity and protein kinase activity et al. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, distinct pathway contained signaling molecules common to various inflammatory and fibrotic pathways were Pathways in cancer, Cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, TNF signaling pathway, MAPK signaling pathway and Toll-like receptor signaling pathway. The BMFBs was treated with heat-inactivated S. aureus (106 CFU/mL) and also with pharmacological inhibitors of ERK1/2, P38 MAPK and JNK. The MMP-2 activity were examined gelatin zymography, MMP-2, TIMP-1, -2 and PLAU/PAI-1 protein expression were examined in vitro by western blot. The MMP-2 activity was significantly inhibited by simultaneous inhibition of ERK1/2, P38 MAPK and JNK, and MMP-2, TIMP-1,-2 and PLAU/PAI-1 protein expression were significantly decreased by inhibiting ERK1/2, P38 MAPK or JNK. This suggested a crosstalk between the ERK1/2, P38 MAPK or JNK signaling pathways in regulating extracellular matrix metabolism in the BMFBs with S. aureus. Our study complement our initial study on S. aureus-induced responses by fibrosis-associated genes in BMFBs. This may lead to development of novel therapeutic targets to control bovine mammary fibrosis induced by S. aureus.