Investigation of the biological activities and metabolomic profiling of thymoquinone in an in vitro model of intestinal inflammation

Gastrointestinal System (GIS) is an anatomical and functional system that regulates the process of digestion and facilitates the absorption of nutrients in the body. The intestinal epithelium, forming a cellular barrier throughout the GIS, is crucial for maintaining mucosal hemostasis. In addition to facilitating nutrient absorption, the intestinal epithelium influences immunity by producing mucus, antimicrobial peptides, and secretory IgA. However, under certain conditions, the normal functions of the intestines can be affected by inflammation, a process that, when balanced, protects the organism from tissue damage in response to harmful agents or injuries. Inflammation is the body's defense mechanism against harmful agents or injuries. Nevertheless, chronic inflammation (prolonged and excessive inflammation) can adversely affect the GIS. Chronic inflammation in the intestines can lead to various chronic diseases, with inflammatory bowel disease (IBD) being a prominent example. IBD is characterized by chronic and recurrent inflammation in the gastrointestinal system. Although an increase in reactive oxygen species/reactive nitrogen species (ROS/RNS) and activation of the NF-κB pathway have been implicated as causes of inflammation, the pathogenesis of IBD is not fully understood. Genetic predisposition is also associated with IBD, and it is a disease with periods of remission, flare-ups, extra-intestinal symptoms, and long-term malignancy risk. The molecular pathogenesis and biomarker determination of IBD have been the focus of numerous metabolomic studies in recent years. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and mass spectrometry (MS), analytical chemistry techniques, are frequently used in metabolomic studies to characterize the metabolites of tissues, biological fluids, or cell extracts qualitatively and quantitatively. The treatment of IBD currently involves drugs such as corticosteroids aimed at controlling inflammation, but these medications have serious side effects. Budesonide, a corticosteroid used in the current thesis, has been shown to control intestinal inflammation but is associated with adverse effects such as immunosuppression and osteoporosis. The search for agents with a lower side effect profile has led to the investigation of polyphenols, known for their antioxidant, anti-inflammatory, and immunomodulatory properties, as potential therapeutic options for inflammatory diseases, including IBD. Thymoquinone (TQ), a polyphenolic compound and the main component of Nigella sativa (black seed), has demonstrated significant antioxidant and anti-inflammatory properties in previous studies. The aim of this study is to investigate the anti-inflammatory therapeutic efficacy of TQ and to profile the disease metabolomics using high-resolution NMR spectroscopy and LC-MS/MS by establishing an in vitro model of intestinal inflammation through cell culture. This research also aims to compare the effects of TQ with budesonide, an anti-inflammatory agent. To create the in vitro model of intestinal inflammation, inflammation was induced in Raw 264.7 macrophage cells by administering lipopolysaccharide (LPS). Subsequently, the inflamed medium was transferred to HT-29 colon cancer cells. The effects of TQ and budesonide on this model were then examined. Reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are key mediators in the development of inflammation, were significantly elevated in the inflammation group. Treatment with TQ resulted in a reduction in the levels of these species, with the most significant decrease observed in the budesonide group. Furthermore, the expression levels of tight junction proteins, occludin and claudin-5, which play a crucial role in the pathogenesis of IBD, increased in the TQ groups, with the highest increase observed in the budesonide group. These results indicate the anti-inflammatory and tissue-healing effects of TQ. Metabolomic analysis using NMR revealed a significant decrease in the xanthine metabolite in the inflammation group compared to the control group, suggesting an impact on the purine metabolic pathway. High-resolution mass spectrometry (HRMS) confirmed this result, indicating the affected pathway. Additionally, several metabolites showed altered levels during inflammation, including lactate, galactarate, fumarate, and dimethylamine, which increased, and glycolate, 2-hydroxyisobutyrate, glycine, acetate, and citraconate, which decreased in the inflammation group compared to the control group. These metabolites are indicative of metabolic changes associated with inflammation. The increase in lactate suggests a shift towards aerobic glycolysis due to the Warburg effect during inflammation. The increase in dimethylamine, a degradation product of asymmetric dimethylarginine (ADMA), implies that the inhibition of nitric oxide synthase (NOS) due to increased ADMA degradation is alleviated during inflammation, contributing to its development. The elevated levels of glycine, glycolate, and citraconate in the control group suggest their roles as antioxidant intermediates in glutathione synthesis, activators of the antioxidant system through serine and glycine, and potent Nrf2 activators, respectively. NMR analysis of the inflammation group treated with TQ revealed a significant decrease in N-nitrosodimethylamine (NDMA), known for its genotoxic and apoptotic properties, compared to the inflammation group. However, no significant metabolic pathway was identified in the inflammation group compared to the control group using NMR analysis, while HRMS indicated a significant impact on the nicotinate-nicotinamide metabolism and ether-lipid metabolism. Budesonide treatment affected only these pathways compared to the inflammation group without inducing inflammation, suggesting that TQ may exert its anti-inflammatory effects through these pathways. In conclusion, the current study employed a combined NMR and MS approach for the comprehensive metabolomic profiling of an in vitro model of intestinal inflammation. The analysis shed light on the potential metabolic pathways through which TQ may exert its anti-inflammatory effects. Furthermore, distinct metabolites that varied during inflammation were identified, laying the groundwork for biomarker studies using cell culture. The similarity of the metabolite profiles obtained from cell culture to those from previous metabolomic studies in IBD patients strengthens the utility of cell culture in metabolomic analyses. However, further in vivo studies are necessary to validate the results obtained from cell culture, as the outcomes may not fully reflect human metabolism. Keywords: inflammation, thymoquinone, metabolomics, cell culture