Giorgis Isaac

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease driven by hepatocellular lipid overload, immune activation, and hepatic stellate cell (HSC)-driven fibrogenesis. Human genetic studies reveal that loss-of-function (LoF) variants in 17 beta-hydroxysteroid dehydrogenase 13 ( HSD17B13 ) confer robust protection against advanced fibrosis and cirrhosis, establishing HSD17B13 as a critical genetic modifier of MASH severity. Yet the mechanisms linking HSD17B13 activity to fibrogenic progression remain poorly understood. Here, we show that both wild-type and catalytically deficient HSD17B13 (mHSD) localize to lipid droplets (LDs) in cultured human hepatocytes, but only catalytically active HSD17B13 enhances hepatocellular lipid accumulation and markedly upregulates the lipogenic transcriptional regulator carbohydrate-responsive element-binding protein (ChREBP). This HSD17B13-driven lipogenic axis elicits potent paracrine activation of LX2 stellate cells, both in hepatocyte-HSC co-culture and in response to hepatocyte-conditioned medium (CM). Screening of candidate signaling mediators revealed that transforming growth factor beta-1 (TGFb-1) is uniquely and strongly upregulated by active HSD17B13, with minimal induction by mHSD. Remarkably, siRNA-mediated knockdown of TGFB1 or neutralization of active TGFb-1 protein abolishes CM-induced LX2 activation and collagen synthesis. Collectively, these findings identify HSD17B13 as a dual metabolic and profibrotic effector that drives TGFb-1-dependent HSC activation, thereby linking hepatocellular lipid dysregulation to fibrogenic progression and providing a mechanistic framework for understanding how HSD17B13 contributes to MASH pathogenesis.

Lipids play an important role in the energy storage, cellular signaling, and pathophysiology of diseases such as cancer, neurodegenerative diseases, infections, and diabetes. Due to high importance of diverse lipid classes in human health and disease, manipulating lipid abundance and composition is an important target for metabolic engineering. The extreme structural diversity of lipids in real biological samples is challenging for analytical techniques due to large difference in physicochemical properties of individual lipid species. This chapter describes lipidomic analysis of large sample sets requiring reliable and robust methodology. Rapid and robust methods facilitate the support of longitudinal studies allowing the transfer of methodology between laboratories. We describe a high-throughput reversed-phase LC-MS methodology using Ultra Performance Liquid Chromatography (UPLC^®) with charged surface hybrid technology and accurate mass detection for high-throughput non-targeted lipidomics. The methodology showed excellent specificity, robustness, and reproducibility for over 100 LC-MS injections.

Within non-communicable diseases, chronic inflammatory conditions represent one of the biggest challenges for modern medicine. Traditional Chinese Medicine (TCM) has been practiced over centuries and has accumulated tremendous empirical knowledge on the treatment of such diseases. Huangqi Jianzhong Tang (HQJZT) is a famous TCM herbal formula composed of Radix Astragali, Ramulus Cinnamomi, Radix et Rhizoma Glycyrrhizae Praeparata cum Melle, Radix Paeoniae Alba, Rhizoma Zingiberis Recens, Fructus Jujubae and Saccharum Granorum (maltose), which has been used for the treatment of various chronic inflammatory gastrointestinal diseases. However, there is insufficient knowledge about its active constituents and the mechanisms responsible for its effects. The present study aimed at identifying constituents contributing to the bioactivity of HQJZT by combining in vitro cytokine production assays and LC-MS metabolomics techniques. From the HQJZT decoction as well as from its single herbal components, extracts of different polarities were prepared. Phytochemical composition of the extracts was analyzed by means of UPLC-QTOF-MS/MS. The inhibitory effects of the extracts on TNF-α, IL-1β and IFN-γ production were studied in U937 cells. Phytochemical and pharmacological bioactivity data were correlated by orthogonal projection to latent structures discriminant analysis (OPLS-DA) in order to identify those HQJZT constituents which may be relevant for the observed pharmacological activities. The investigations resulted in the identification of 16 HQJZT constituents, which are likely to contribute to the activities observed in U937 cells. Seven of them, namely calycosin, formononetin, astragaloside I, liquiritigenin, 18β-glycyrrhetinic acid, paeoniflorin and albiflorin were unambiguously identified. The predicted results were verified by testing these compounds in the same pharmacological assays as for the extracts. In conclusion, the anti-inflammatory activity of HQJZT could be substantiated by in vitro pharmacological screening, and the predicted activities of the OPLS-DA hits could be partially verified. Moreover, the benefits and limitations of MVDA for prediction pharmacologically active compounds contributing to the activity of a TCM mixture could be detected.

Eleutherococcus senticosus Maxim. belongs to the Araliaceae family. Phytochemical studies reveal that E. senticosus leaves contain triterpene glycosides along with organic acid derivatives and flavonoid compounds. It is believed that E. senticosus is similar to ginseng because they come from same family and both contain triterpene saponins. E. senticosus leaves have been developed as a functional beverage called ci-wu-jia tea in recent years. Triterpene glycosides are difficult to identify by ultraviolet (UV) detection and contents of these compounds are low in E. senticosus leaves. In this study, a sensitive ultra-high performance liquid chromatographic (UHPLC) method combining UV and tandem mass spectrometry (MS/MS) was developed to characterize the triterpene glycosides from E. senticosus leaves and related commercial products. Fragmentation patterns of three sub-groups of triterpene glycosides in E. senticosus leaves were investigated. Additionally, fragmentation pathways and UV characteristics of organic acid derivatives and flavonoids were also characterized. A compound screening library, including 241 compounds reported in the literature, was created and used to confirm the compounds in the samples. In this study, a total of 24 samples, including 13 plant samples of E. senticosus and 11 ci-wu-jia tea products, were analyzed. Out of the 11 commercial products, three products were discovered to contain green tea (Camellia sinensis) that was considered to be an adulterant since it was not an ingredient on the labels. The developed UHPLC-UV-MS/MS analytical method combined with the UNIFI processing method can simultaneously characterize organic acid derivatives, flavonoids, and triterpene saponins from E. senticosus. It provides a simple and sensitive way to perform quality control of E. senticosus and related ci-wu-jia tea products.

Ultra-high performance supercritical fluid chromatography (UHPSFC) is a separation technique that provides an increase in selectivity, efficiency and sensitivity. UHPSFC uses compressed CO2 as the primary mobile phase to leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease of use and method development simplicity of normal-phase LC. The use of CO2 as primary solvent results in significant savings in solvent costs and solvent waste disposal. The miscibility of CO2 with a wide range of polar and non-polar organic solvents has made the liquid CO2 based mobile phase versatile enough to separate a much wider variety of natural product compounds. UHPSFC in combination with sub-2 µm particles and either diode array ultraviolet, evaporative light scattering, or mass spectrometric detection has been shown to be a valuable technique for the analysis of natural products. In this study, sub-2 µm particle CO2 based UHPSFC was investigated for the separation and analysis of different natural products including terpene lactones and ginkgolic acids in Ginkgo biloba, cannabinoids in cannabis and cannabis products, volatiles in traditional medicine, beta-carotene, lipids and other compounds in different extracts. The method development and optimization involved the investigation of different columns, modifiers and instrument conditions. The benefit of UHPSFC will be highlighted and discussed compared to the traditional gas chromatography and normal phase chromatography for natural product analysis. For most of the applications better separation and selectivity was obtained with significant reduction in analysis time compared to alternative techniques such as GC which requires derivatization.

Natural products chemical ingredient profiling is a challenging task because of the sample complexity and the analyses required. For any natural products related study, regardless of the research focus and the final end goal, ingredient profiling (to elucidate the active compounds from a particular plant), sample comparison (to understand point of origin and to prove authenticity), quantification and identification of target compounds for quality control purpose are all routinely required. Because of the complexity of the sample and large amount of data that can be generated using the most popular approach such as LC/MS, researchers are still challenged by how to narrow down the scope and to obtain meaningful information quickly. Three different botanicals (Hoodia, Terminalia and Chamomile) and their respective dietary supplements were used for the study of targeted and non-targeted analysis of compounds. Here, a streamlined workflow was presented that enables researchers to quickly identify chemical ingredients from botanicals and dietary supplements from a single data independent LC/MS injection. The data independent acquisition provides both precursor exact mass and corresponding fragment ion information from a single injection for facile structural characterization. Data are then searched against the Traditional Medicine Scientific Library provided within the informatics platform from the instrument vendor. Structures of matched experimental and in silico corresponding fragment ions are automatically verified by a software tool imbedded within UNIFI informatics platform. Results are displayed automatically using preset workflow and report templates.