Application of Systems Biology Analysis to Hepatic Injury Following Hemorrhagic Shock

Abstract

<bold>Introduction:</bold> This dissertation is focused on the metabolomic and transcriptomic changes that occur as a result of carbohydrate prefeeding during hemorrhagic shock and trauma within the liver of a porcine model. The risk of trauma and hemorrhagic shock continues to be an important issue in both military and civilian sectors. As such, we explored the impact of a prior fed state upon the overall response to hemorrhagic shock and resuscitation. The primary hypotheses were that changes in metabolism at the metabolomic and transcriptomic levels would be dependent upon the fed state. In addition, this thesis explores a more comprehensive analysis of metabolomics datasets to standardize analysis and improve overall consistency.<bold>Materials and Methods:</bold> Algorithm comparison was accomplished using six commonly applied methods to three synthetic datasets, of different sample sizes, and three openly accessible published datasets. This comparison also incorporated metrics to measure consistency of identified features (i.e. stability) to provide further confidence in results. Metabolomics analysis was accomplished with nuclear magnetic resonance spectroscopy (NMR) and Chenomx software to profile and quantify metabolites in liver extracts. The metabolome was subsequently analyzed with partial least squares discriminant analysis (PLS-DA). Transcriptomics analysis was conducted using next-generation sequencing (NGS) technology to employ RNA-sequencing (RNA-seq) on mRNA extracts from liver biopsies. The RNA-seq data was analyzed using typical processing techniques to generate a count matrix and subsequently analyzed with the Bioconductor package EdgeR. Results:</bold> The comparison of algorithms showed that the best algorithm is associated with differently structured datasets (e.g. number of features, number of groups, sample size, etc.). Analysis of the liver metabolome revealed changes in carbon energy sources, amino acid metabolism, oxidative stress, and membrane maintenance. Transcriptomic analysis revealed changes in carbohydrate metabolism, cytokine inflammation, cholesterol synthesis and apoptosis. In addition, there is evidence of increased cytoskeleton reorganization which may correspond to a shrunken, catabolic state which provides and anti-inflammatory condition to mitigate cellular damage.<bold>Conclusion:</bold> The response to hemorrhagic shock and resuscitation is altered with respect to a fasted or carbohydrate prefed state. Metabolomics and transcriptomic analyses suggest altered metabolic pathways as a result of fed state. Altered carbohydrate metabolism was readily identified thereby confirming both methods were successful. Additionally, indications of membrane maintenance that follow cytoskeletal remodeling and cellular shrinkage are potentially reflected by 3-Hydroxyisovalerate and sn-Glycero-3-phosphocholine. These results provide further evidence for pre-conditioning (e.g. altered diet) and hypertonic resuscitation methods to possibly improve patient outcome. Further research is required in alternative prefeeding substrates (e.g. protein, lipid, etc.) as well as improving the integration of different systems level datasets to understand more thoroughly the systemic effects of hemorrhagic shock and resuscitation.