Maria Wallert

Background: Metabolic dysfunction-associated steatohepatitis (MASH) represents a critical progression of metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by hepatic steatosis, inflammation, and beginning fibrosis. The selection of appropriate animal models is crucial for understanding disease mechanisms and evaluating therapeutic interventions. While wild-type C57BL/6 mice are widely used, hyperlipidemic apolipoprotein E-deficient ( Apoe ^-/- ) mice may offer superior disease modeling capabilities. This study compared the development and progression of diet-induced MASH in Apoe ^-/- mice versus wild-type C57BL/6J mice over multiple time points, with a focus on inflammatory responses and histological changes. Methods: Male wild-type C57BL/6J and male Apoe ^-/- mice were fed either standard chow diet or high-fat/high-fructose (Hf/Hf) diet for 8, 12, 16, or 24 weeks. Comprehensive analyses included histological evaluation, MRS based fat quantification, immunohistochemistry, serum biochemistry, cytokine profiling, trace element analysis, oxylipin profiling by UPLC-MS/MS, intestinal barrier function assessment, and gene expression analysis. Results: Apoe ^-/- mice showed markedly accelerated MASH development compared to wild-type mice. Notably, Apoe ^-/- mice fed Hf/Hf diet for 12 weeks exhibited substantially greater inflammatory infiltration than wild-type mice even after 24 weeks of Hf/Hf diet. This enhanced inflammatory response was accompanied by increased infiltration of macrophages (CD68 ⁺ cells), elevated levels of pro-inflammatory cytokines, altered trace element homeostasis, and dysregulated oxylipin profiles favoring pro-inflammatory mediators. There was also evidence of compromised intestinal barrier function and more pronounced histological features of MASH, including steatosis, lobular inflammation and hepatocyte ballooning. Conclusion: Apoe ^-/- mice represent a superior model for MASH research, demonstrating accelerated disease progression and enhanced inflammatory responses compared to the wild-type mice. The rapid and robust inflammatory phenotype in Apoe ^-/- mice provides a more efficient and clinically relevant model for investigating MASH pathogenesis and testing therapeutic interventions.

The accurate definition of in vivo , ex vivo and in vitro models is critical for the whole R&D process, i.e., basic research, clinical translation and reliability of results. Although many models are currently being developed, it is important to recognize the limitations and advantages of each of them. The aim of this review is to compile the most important alternatives to animal models in the fields of gastroenterology and hepatology research. A thorough comparison and understanding of each alternative model will certainly save time and money and will lead to better predictability and reliability of results for clinical translation and clinical trials. There is no single model capable of replacing the complexity of human biology. Nevertheless, it is essential to understand the fundamentals of human anatomy, physiology and disease pathophysiology to select the most appropriate model in translational research. At the same time, the appropriate model must be selected to gain a deeper understanding of the principal processes underlying human physiology and the pathology of diseases.

RAW264.7 cells are a macrophage-like cell line that was derived from a male BALB/c mouse ascites tumor induced by Abelson Leukemia Virus (A-MuLV) [1]. Due to its rapid proliferation and ease of handling, the RAW264.7 cell line is widely used to study immune function, inflammation, and cancer biology. The cells have a doubling time of about 11 to 30 hours. The growth media should be refreshed every 2 to 3 days but not exceeding 4 days. RAW264.7 cells can perform phagocytosis and pinocytosis, which are essential functions of macrophages. In addition, RAW264.7 M0 macrophages can differentiate into M1 or M2 macrophages upon respective stimuli. Hence, stimulated RAW264.7 cells have also been widely used as an in vitro inflammatory model for screening the effectivity of anti-inflammatory compounds and the investigation of the underlying molecular mechanism.

THP-1 is a human monocytic cell line originally isolated from a 1-year old male subject with acute monocytic leukemia (Tsuchiya et al., 1980). The cells are easy to culture and are safe, as no viruses have been reported to be present in the cells (Chanput et al., 2014). THP-1 monocytes are round, floating cells with a doubling time of 35-50 h (Chanput et al., 2014). They have been used as a model for studying immune response, monocyte and macrophage function, lipid metabolism and functions of drugs (Chanput et al., 2014). THP-1 cells can be differentiated into macrophages for use in experiments. As macrophages, the cells become adherent to the cell culture vessel. Agents used for differentiation of THP-1 cells include phorbol 12-myristate 13-acetate (PMA), vitamin D, and macrophage colony-stimulation factor M-CSF (Chanput et al., 2014). This cell line is sensitive to culture conditions; e.g. the cells react differently to differentiation substances depending on the cell density (Liu et al., 2023). The protocol presented herein is routinely used for culturing and differentiating THP-1 cells with PMA.

Excessive inflammation in cells are a common cause of inflammation-related diseases such as cardiometabolic diseases. The cellular multiprotein complex nucleotide-binding domain and leucine-rich repeat pyrin domain 3 (NLRP3) inflammasome is a cellular key modulator of inflammatory processes. In addition to classic medications, phytochemicals are known for their anti-inflammatory potential. In African folk medicine the seeds of Garcinia kola are used to support the treatment of inflammatory diseases. Of particular interest is the phytochemical garcinoic acid (GA, trans -13′-carboxy-δ-tocotrienol), which is isolated from the Garcinia kola seeds. This derivative and potential metabolite of the vitamin E congener δ-tocotrienol (T3) shows anti-inflammatory properties in vitro. However, the underlying mechanisms are largely unknown. To get better insights into the molecular mode of action, murine J774A.1 macrophages were stimulated with lipopolysaccharides (LPS) only or in combination with adenosine triphosphate (ATP), which led to canonical activation of the NLRP3 inflammasome and subsequent pyroptosis. A combined treatment with GA resulted in significantly reduced stimulation of the transcription factor nuclear factor 'ĸ-light-chain-enhancer' of activated B-cells (NF-ĸB), decreased expression of inflammasome-related genes and marked downregulation of autoproteolytic cleavage of caspase-1 (Casp-1). Consequently, GA had an inhibitory effect on pyroptosis. The results have been validated using the well-known NLRP3 inflammasome inhibitor MCC950. In conclusion, GA was shown to have relevant effects on the regulation of the NLRP3 inflammasome and pyroptosis in vitro. Our study provides new mechanistic insights into the anti-inflammatory mode of action of GA and highlights its relevance as a potential phytochemical drug for the treatment of inflammation.

HepG2 cells, derived from the liver tissue of a 15-year-old male patient with hepatocellular carcinoma [1], are a widely used cell line for in vitro studies of liver metabolism, toxicology and drug testing [2]. These cells have a doubling time of approximately 48 hours and can be maintained for up to 25 passages after thawing, while retaining their characteristics [1,3]. However, despite their widespread use, HepG2 cells do not retain all the characteristics of primary human hepatocytes. Although they express some metabolic enzymes, such as members of the cytochrome P450 family, their enzyme activity is significantly lower than that of primary hepatocytes [2,4]. This limitation must be taken into account when interpreting pharmacokinetic and toxicity data.

THP-1 is a human monocytic cell line originally isolated from a 1-year old male subject with acute monocytic leukemia (Tsuchiya et al., 1980). The cells are easy to culture and are safe, as no viruses have been reported to be present in the cells (Chanput et al., 2014). THP-1 monocytes are round, floating cells with a doubling time of 35-50 h (Chanput et al., 2014). They have been used as a model for studying immune response, monocyte and macrophage function, lipid metabolism and functions of drugs (Chanput et al., 2014). THP-1 cells can be differentiated into macrophages for use in experiments. As macrophages, the cells become adherent to the cell culture vessel. Agents used for differentiation of THP-1 cells include phorbol 12-myristate 13-acetate (PMA), vitamin D, and macrophage colony-stimulation factor M-CSF (Chanput et al., 2014). This cell line is sensitive to culture conditions; e.g. the cells react differently to differentiation substances depending on the cell density (Liu et al., 2023). The protocol presented herein is routinely used for culturing and differentiating THP-1 cells with PMA.

The mouse model of 2,4-dinitrochlorbenzene (DNCB)-induced human-like atopic dermatitis (hlAD) has been widely used to test novel treatment strategies and compounds. However, the study designs and methods are highly diverse, presenting different hlAD disease patterns that occur after sensitization and repeated challenge with DNCB on dorsal skin. In addition, there is a lack of information about the progression of the disease during the experiment and the achieved pheno- and endotypes, especially at the timepoint when therapeutic treatment is initiated. We here examine hlAD in a DNCB-induced BALB/cJRj model at different timepoints: (i) before starting treatment with dexamethasone, representing a standard drug control (day 12) and (ii) at the end of the experiment (day 22). Both timepoints display typical AD-associated characteristics: skin thickening, spongiosis, hyper- and parakeratosis, altered cytokine and gene expression, increased lipid mediator formation, barrier protein and antimicrobial peptide abnormalities, as well as lymphoid organ hypertrophy. Increased mast cell infiltration into the skin and elevated immunoglobulin E plasma concentrations indicate a type I allergy response. The DNCB-treated skin showed an extrinsic moderate sub-acute hlAD lesion at day 12 and an extrinsic mild sub-acute to chronic pheno- and endotype at day 22 with a dominating Th2 response. A dependency of the filaggrin formation and expression in correlation to the disease severity in the DNCB-treated skin was found. In conclusion, our study reveals a detailed classification of a hlAD at two timepoints with different inflammatory skin conditions and pheno- and endotypes, thereby providing a better understanding of the DNCB-induced hlAD model in BALB/cJRj mice.