Liver-on-chip model and application in predictive genotoxicity and mutagenicity of drugs.

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.

Modeling alcohol-associated liver disease in humans using adipose stromal or stem cell-derived organoids.

Alcohol-associated liver disease (ALD) is a prevalent liver disease, yet research is hampered by the lack of suitable and reliable human ALD models. Herein, we generated human adipose stromal/stem cell (hASC)-derived hepatocellular organoids (hAHOs) and hASC-derived liver organoids (hALOs) in a three-dimensional system using hASC-derived hepatocyte-like cells and endodermal progenitor cells, respectively. The hAHOs were composed of major hepatocytes and cholangiocytes. The hALOs contained hepatocytes and nonparenchymal cells and possessed a more mature liver function than hAHOs. Upon ethanol treatment, both steatosis and inflammation were present in hAHOs and hALOs. The incubation of hALOs with ethanol resulted in increases in the levels of oxidative stress, the endoplasmic reticulum protein thioredoxin domain-containing protein 5 (TXNDC5), the alcohol-metabolizing enzymes ADH1B and ALDH1B1, and extracellular matrix accumulation, similar to those of liver tissues from patients with ALD. These results present a useful approach for understanding the pathogenesis of ALD in humans, thus facilitating the discovery of effective treatments.

HSDL2 links nutritional cues to bile acid and cholesterol homeostasis.

In response to energy and nutrient shortage, the liver triggers several catabolic processes to promote survival. Despite recent progress, the precise molecular mechanisms regulating the hepatic adaptation to fasting remain incompletely characterized. Here, we report the identification of hydroxysteroid dehydrogenase-like 2 (HSDL2) as a mitochondrial protein highly induced by fasting. We show that the activation of PGC1α-PPARα and the inhibition of the PI3K-mTORC1 axis stimulate HSDL2 expression in hepatocytes. We found that HSDL2 depletion decreases cholesterol conversion to bile acids (BAs) and impairs FXR activity. HSDL2 knockdown also reduces mitochondrial respiration, fatty acid oxidation, and TCA cycle activity. Bioinformatics analyses revealed that hepatic Hsdl2 expression positively associates with the postprandial excursion of various BA species in mice. We show that liver-specific HSDL2 depletion affects BA metabolism and decreases circulating cholesterol levels upon refeeding. Overall, our report identifies HSDL2 as a fasting-induced mitochondrial protein that links nutritional signals to BAs and cholesterol homeostasis.

METTL3 Deficiency Aggravates Hepatic Ischemia/Reperfusion Injury in Mice by Activating the MAPK Signaling Pathway.

Background: Inflammatory responses, apoptosis, and oxidative stress, are key factors that contribute to hepatic ischemia/reperfusion (I/R) injury, which may lead to the failure of liver surgeries, such as hepatectomy and liver transplantation. The N6-methyladenosine (m6A) modification has been implicated in multiple biological processes, and its specific role and mechanism in hepatic I/R injury require further investigation. Methods: Dot blotting analysis was used to profile m6A levels in liver tissues at different reperfusion time points in hepatic I/R mouse models. Hepatocyte-specific METTL3 knockdown (HKD) mice were used to determine the function of METTL3 during hepatic I/R. RNA sequencing and western blotting were performed to assess the potential signaling pathways involved with the deficiency of METTL3. Finally, AAV8-TBG-METTL3 was injected through the tail vein to further elucidate the role of METTL3 in hepatic I/R injury. Results: The m6A modification levels and the expression of METTL3 were upregulated in mouse livers during hepatic I/R injury. METTL3 deficiency led to an exacerbated inflammatory response and increased cell death during hepatic I/R, whereas overexpression of METTL3 reduced the extent of liver injury. Bioinformatic analysis revealed that the MAPK pathway was significantly enriched in the livers of METTL3-deficient mice. METTL3 protected the liver from I/R injury, possibly by inhibiting the phosphorylation of JNK and ERK, but not P38. Conclusions: METTL3 deficiency aggravates hepatic I/R injury in mice by activating the MAPK signaling pathway. METTL3 may be a potential therapeutic target in hepatic I/R injury.

Hepatoprotective effects of baicalein against liver ischemia-reperfusion injury and partial hepatectomy in a rat model.

BACKGROUND: Baicalein is the main active flavonoid in Scutellariae Radix and is included in shosaikoto, a Kampo formula used for treating hepatitis and jaundice. However, little is known about its hepatoprotective effects against hepatic ischemia-reperfusion injury (HIRI), a severe clinical condition directly caused by interventional procedures. We aimed to investigate the hepatoprotective effects of baicalein against HIRI and partial hepatectomy (HIRI + PH) and its potential underlying mechanisms. METHODS AND RESULTS: Male Sprague-Dawley rats received either baicalein (5 mg/kg) or saline intraperitoneally and underwent a 70% hepatectomy 15 min after hepatic ischemia. After reperfusion, liver and blood samples were collected. Survival was monitored 30 min after hepatic ischemia and hepatectomy. In interleukin 1ß (IL-1ß)-treated primary cultured rat hepatocytes, the influence of baicalein on inflammatory mediator production and the associated signaling pathway was analyzed. Baicalein suppressed apoptosis and neutrophil infiltration, which are the features of HIRI + PH treatment-induced histological injury. Baicalein also reduced the mRNA expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). In addition, HIRI + PH treatment induced liver enzyme deviations in the serum and hypertrophy of the remnant liver, which were suppressed by baicalein. In the lethal HIRI + PH treatment group, baicalein significantly reduced mortality. In IL-1ß-treated rat hepatocytes, baicalein suppressed TNF-α and chemokine mRNA expression as well as the activation of nuclear factor-kappa B (NF-κB) and Akt. CONCLUSIONS: Baicalein treatment attenuates HIRI + PH-induced liver injury and may promote survival. This potential hepatoprotection may be partly related to suppressing inflammatory gene induction through the inhibition of NF-κB activity and Akt signaling in hepatocytes.

3D microscaffolds with triple-marker sensitive nanoprobes for studying fatty liver disease in vitro.

Non-alcoholic fatty liver disease (NAFLD) is a heterogeneous condition that encompasses a wide range of liver diseases that progresses from simple hepatic steatosis to the life-threatening state of cirrhosis. However, due to the heterogeneity of this disease, comprehensive analysis of several physicochemical and biological factors that drive its progression is necessary. Therefore, an in vitro platform is required that would enable real-time monitoring of these changes to better understand the progression of these diseases. The earliest stage of NAFLD, i.e. hepatic steatosis, is characterised by triglyceride accumulation in the form of lipid vacuoles in the cytosol of hepatocytes. This fatty acid accumulation is usually accompanied by hepatic inflammation, leading to tissue acidification and dysregulated expression of certain proteases such as matrix metalloproteinases (MMPs). Taking cues from the biological parameters of the disease, we report here a 3D in vitro GelMA/alginate microscaffold platform encapsulating a triple-marker (pH, MMP-3 and MMP-9) sensitive fluorescent nanoprobe for monitoring, and hence, distinguishing the fatty liver disease (hepatic steatosis) from healthy livers on the basis of pH change and MMP expression. The nanoprobe consists of a carbon nanoparticle (CNP) core, which exhibits intrinsic pH-dependent fluorescence properties, decorated either with an MMP-3 (NpMMP3) or MMP-9 (NpMMP9) sensitive peptide substrate. These peptide substrates are flanked with a fluorophore-quencher pair that separates on enzymatic cleavage, resulting in fluorescence emission. The cocktail of these nanoprobes generated multiple fluorescence signals corresponding to slightly acidic pH (blue) and overexpression of MMP-3 (green) and MMP-9 (red) enzymes in a 3D in vitro fatty liver model, whereas no/negligible fluorescence signals were observed in a healthy liver model. Moreover, this platform enabled us to mimic fatty liver disease in a more realistic manner. Therefore, this 3D in vitro platform encapsulating triple-marker sensitive fluorescent nanoprobes would facilitate the monitoring of the changes in pH and MMP expression, thereby enabling us to distinguish a healthy liver from a diseased liver and to study liver disease stages on the basis of these markers.

Glycyrrhetinic Acid as a Hepatocyte Targeting Ligand-Functionalized Platinum(IV) Complexes for Hepatocellular Carcinoma Therapy and Overcoming Multidrug Resistance.

Promising targeted therapy options to overcome drug resistance and side effects caused by platinum(II) drugs for treatment in hepatocellular carcinoma are urgently needed. Herein, six novel multifunctional platinum(IV) complexes through linking platinum(II) agents and glycyrrhetinic acid (GA) were designed and synthesized. Among them, complex 20 showed superior antitumor activity against tested cancer cells including cisplatin resistance cells than cisplatin and simultaneously displayed good liver-targeting ability. Moreover, complex 20 can significantly cause DNA damage and mitochondrial dysfunction, promote reactive oxygen species generation, activate endoplasmic reticulum stress, and eventually induce apoptosis. Additionally, complex 20 can effectively inhibit cell migration and invasion and trigger autophagy and ferroptosis in HepG-2 cells. More importantly, complex 20 demonstrated stronger tumor inhibition ability than cisplatin or the combo of cisplatin/GA with almost no systemic toxicity in HepG-2 or A549 xenograft models. Collectively, complex 20 could be developed as a potential anti-HCC agent for cancer treatment.

Binucleated human hepatocytes arise through late cytokinetic regression during endomitosis M phase.

Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.

Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats.

Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.

Efficient hepatocyte differentiation of primary human hepatocyte-derived organoids using three dimensional nanofibers (HYDROX) and their possible application in hepatotoxicity research.

Human liver organoids are in vitro three dimensionally (3D) cultured cells that have a bipotent stem cell phenotype. Translational research of human liver organoids for drug discovery has been limited by the challenge of their low hepatic function compared to primary human hepatocytes (PHHs). Various attempts have been made to develop functional hepatocyte-like cells from human liver organoids. However, none have achieved the same level of hepatic functions as PHHs. We here attempted to culture human liver organoids established from cryopreserved PHHs (PHH-derived organoids), using HYDROX, a chemically defined 3D nanofiber. While the proliferative capacity of PHH-derived organoids was lost by HYDROX-culture, the gene expression levels of drug-metabolizing enzymes were significantly improved. Enzymatic activities of cytochrome P450 3A4 (CYP3A4), CYP2C19, and CYP1A2 in HYDROX-cultured PHH-derived organoids (Org-HYDROX) were comparable to those in PHHs. When treated with hepatotoxic drugs such as troglitazone, amiodarone and acetaminophen, Org-HYDROX showed similar cell viability to PHHs, suggesting that Org-HYDROX could be applied to drug-induced hepatotoxicity tests. Furthermore, Org-HYDROX maintained its functions for up to 35 days and could be applied to chronic drug-induced hepatotoxicity tests using fialuridine. Our findings demonstrated that HYDROX could possibly be a novel biomaterial for differentiating human liver organoids towards hepatocytes applicable to pharmaceutical research.

Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

Liver-specific mitochondrial amidoxime-reducing component 1 (Mtarc1) knockdown protects the liver from diet-induced MASH in multiple mouse models.

BACKGROUND: Human genetic studies have identified several mitochondrial amidoxime-reducing component 1 (MTARC1) variants as protective against metabolic dysfunction-associated steatotic liver disease. The MTARC1 variants are associated with decreased plasma lipids and liver enzymes and reduced liver-related mortality. However, the role of mARC1 in fatty liver disease is still unclear. METHODS: Given that mARC1 is mainly expressed in hepatocytes, we developed an N-acetylgalactosamine-conjugated mouse Mtarc1 siRNA, applying it in multiple in vivo models to investigate the role of mARC1 using multiomic techniques. RESULTS: In ob/ob mice, knockdown of Mtarc1 in mouse hepatocytes resulted in decreased serum liver enzymes, LDL-cholesterol, and liver triglycerides. Reduction of mARC1 also reduced liver weight, improved lipid profiles, and attenuated liver pathological changes in 2 diet-induced metabolic dysfunction-associated steatohepatitis mouse models. A comprehensive analysis of mARC1-deficient liver from a metabolic dysfunction-associated steatohepatitis mouse model by metabolomics, proteomics, and lipidomics showed that Mtarc1 knockdown partially restored metabolites and lipids altered by diet. CONCLUSIONS: Taken together, reducing mARC1 expression in hepatocytes protects against metabolic dysfunction-associated steatohepatitis in multiple murine models, suggesting a potential therapeutic approach for this chronic liver disease.

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth.

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

A patient-based iPSC-derived hepatocyte model of alcohol-associated cirrhosis reveals bioenergetic insights into disease pathogenesis.

Only ~20% of heavy drinkers develop alcohol cirrhosis (AC). While differences in metabolism, inflammation, signaling, microbiome signatures and genetic variations have been tied to the pathogenesis of AC, the key underlying mechanisms for this interindividual variability, remain to be fully elucidated. Induced pluripotent stem cell-derived hepatocytes (iHLCs) from patients with AC and healthy controls differ transcriptomically, bioenergetically and histologically. They include a greater number of lipid droplets (LDs) and LD-associated mitochondria compared to control cells. These pre-pathologic indicators are effectively reversed by Aramchol, an inhibitor of stearoyl-CoA desaturase. Bioenergetically, AC iHLCs have lower spare capacity, slower ATP production and their mitochondrial fuel flexibility towards fatty acids and glutamate is weakened. MARC1 and PNPLA3, genes implicated by GWAS in alcohol cirrhosis, show to correlate with lipid droplet-associated and mitochondria-mediated oxidative damage in AC iHLCs. Knockdown of PNPLA3 expression exacerbates mitochondrial deficits and leads to lipid droplets alterations. These findings suggest that differences in mitochondrial bioenergetics and lipid droplet formation are intrinsic to AC hepatocytes and can play a role in its pathogenesis.

Asprosin aggravates nonalcoholic fatty liver disease via inflammation and lipid metabolic disturbance mediated by reactive oxygen species.

Asprosin (ASP) is a newly-identified adipokine and plays important roles in energy metabolism homeostasis. However, there is no report on whether and how ASP is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Therefore, in the study, we investigated the protective effects of ASP-deficiency on the liver in the NAFLD model mice and the detrimental effects of ASP treatment on the human normal hepatocytes (LO2 cell line). More important, we explored the underlying mechanism from the perspective of lipid metabolism and inflammation. In the in vivo experiments, our data showed that the ASP-deficiency significantly alleviated the high-fat diet-induced inflammation and NAFLD, inhibited the hepatic fat deposition and downregulated the expressions of fat acid synthase (FASN), peroxisome proliferator-activated receptor γ (PPARγ) and forkhead box protein O1 (FOXO1); moreover, the ASP-deficiency attenuated the inflammatory state and inhibited the activation of the IKK/NF-κBp65 inflammation pathway. In the in vitro experiments, our results revealed that ASP treatment caused and even exacerbated the injury of LO2 cells induced by FFA; In contrast, the ASP treatment upregulated the expressions of PPARγ, FOXO1, FASN, ACC and acyl-CoA oxidase 1 (ACOX1) and elevated the reactive oxygen species (ROS) levels. Accordingly, these results demonstrate that ASP causes NAFLD through disrupting lipid metabolism and promoting the inflammation mediated by ROS.

Resveralogues protect HepG2 cells against cellular senescence induced by hepatotoxic metabolites.

Progressive liver disease and dysfunction cause toxic metabolites including ammonia and unconjugated bilirubin to accumulate in plasma. As the population ages alternatives to liver transplantation become increasingly important. One approach for use as a bridge to transplant or recovery is the use of bioartificial liver systems (BALS) containing primary or immortalised hepatocytes as ex-vivo replacements or supports for endogenous liver function. However, exposure to the hepatotoxic metabolites present in plasma causes the rapid failure of these cells to carry out their primary metabolic functions despite remaining viable. Hypothesizing that this loss of core hepatocyte phenotypes was caused by cell senescence we exposed HepG2 cell populations, grown in both standard two-dimensional tissue culture systems and in three dimensional cultures on novel alginate modified HEMA-MBA cryogels, to physiologically reflective concentrations of hepatotoxic metabolites and cytokines. HepG2 cells are forced into senescence by the toxic metabolites in under six hours (as measured by loss of thymidine analog incorporation or detectable Ki67 staining) which is associated with a ten to twenty-fold reduction in the capacity of the cultures to synthesise albumin or urea. This state of senescence induced by liver toxins (SILT) can be prevented by preincubation with either 2-5 µM resveratrol, its major in vivo metabolite dihydroresveratrol or a series of novel resveralogues with differential capacities to scavenge radicals and activate SIRT1 (including V29 which does not interact with the protein). SILT appears to be a previously unrecognised barrier to the development of BALS which can now be overcome using small molecules that are safe for human use at concentrations readily achievable in vivo.

Rutin attenuates ensartinib-induced hepatotoxicity by non-transcriptional regulation of TXNIP.

Ensartinib, an approved ALK inhibitor, is used as a first-line therapy for advanced ALK-positive non-small cell lung cancer in China. However, the hepatotoxicity of ensartinib seriously limits its clinical application and the regulatory mechanism is still elusive. Here, through transcriptome analysis we found that transcriptional activation of TXNIP was the main cause of ensartinib-induced liver dysfunction. A high TXNIP level and abnormal TXNIP translocation severely impaired hepatic function via mitochondrial dysfunction and hepatocyte apoptosis, and TXNIP deficiency attenuated hepatocyte apoptosis under ensartinib treatment. The increase in TXNIP induced by ensartinib is related to AKT inhibition and is mediated by MondoA. Through screening potential TXNIP inhibitors, we found that the natural polyphenolic flavonoid rutin, unlike most reported TXNIP inhibitors can inhibit TXNIP by binding to TXNIP and partially promoting its proteasomal degradation. Further studies showed rutin can attenuate the hepatotoxicity of ensartinib without antagonizing its antitumor effects. Accordingly, we suggest that TXNIP is the key cause of ensartinib-induced hepatotoxicity and rutin is a potential clinically safe and feasible therapeutic strategy for TXNIP intervention.

Upregulation of Hepatic Glutathione S-Transferase Alpha 1 Ameliorates Metabolic Dysfunction-Associated Steatosis by Degrading Fatty Acid Binding Protein 1.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common metabolic disease of the liver, characterized by hepatic steatosis in more than 5% of hepatocytes. However, despite the recent approval of the first drug, resmetirom, for the management of metabolic dysfunction-associated steatohepatitis, decades of target exploration and hundreds of clinical trials have failed, highlighting the urgent need to find new druggable targets for the discovery of innovative drug candidates against MASLD. Here, we found that glutathione S-transferase alpha 1 (GSTA1) expression was negatively associated with lipid droplet accumulation in vitro and in vivo. Overexpression of GSTA1 significantly attenuated oleic acid-induced steatosis in hepatocytes or high-fat diet-induced steatosis in the mouse liver. The hepatoprotective and anti-inflammatory drug bicyclol also attenuated steatosis by upregulating GSTA1 expression. A detailed mechanism showed that GSTA1 directly interacts with fatty acid binding protein 1 (FABP1) and facilitates the degradation of FABP1, thereby inhibiting intracellular triglyceride synthesis by impeding the uptake and transportation of free fatty acids. Conclusion: GSTA1 may be a good target for the discovery of innovative drug candidates as GSTA1 stabilizers or enhancers against MASLD.

Finding a Direct Method for a Dynamic Process: The DD (Direct and Dynamic) Cell-Tox Method.

The main focus of in vitro toxicity assessment methods is to assess the viability of the cells, which is usually based on metabolism changes. Yet, when exposed to toxic substances, the cell triggers multiple signals in response. With this in mind, we have developed a promising cell-based toxicity method that observes various cell responses when exposed to toxic substances (either death, division, or remain viable). Based on the collective cell response, we observed and predicted the dynamics of the cell population to determine the toxicity of the toxicant. The method was tested with two different conformations: In the first conformation, we exposed a monoculture model of blood macrophages to UV light, hydrogen peroxide, nutrient deprivation, tetrabromobisphenol A, fatty acids, and 5-fluorouracil. In the second, we exposed a coculture liver model consisting of hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells to rifampicin, ibuprofen, and 5-fluorouracil. The method showed good accuracy compared to established toxicity assessment methods. In addition, this approach provided more representative information on the toxic effects of the compounds, as it considers the different cellular responses induced by toxic agents.

Inhibition of Drp1-Filamin Protein Complex Prevents Hepatic Lipid Droplet Accumulation by Increasing Mitochondria-Lipid Droplet Contact.

Lipid droplet (LD) accumulation in hepatocytes is one of the major symptoms associated with fatty liver disease. Mitochondria play a key role in catabolizing fatty acids for energy production through ß-oxidation. The interplay between mitochondria and LD assumes a crucial role in lipid metabolism, while it is obscure how mitochondrial morphology affects systemic lipid metabolism in the liver. We previously reported that cilnidipine, an already existing anti-hypertensive drug, can prevent pathological mitochondrial fission by inhibiting protein-protein interaction between dynamin-related protein 1 (Drp1) and filamin, an actin-binding protein. Here, we found that cilnidipine and its new dihydropyridine (DHP) derivative, 1,4-DHP, which lacks Ca2+ channel-blocking action of cilnidipine, prevent the palmitic acid-induced Drp1-filamin interaction, LD accumulation and cytotoxicity of human hepatic HepG2 cells. Cilnidipine and 1,4-DHP also suppressed the LD accumulation accompanied by reducing mitochondrial contact with LD in obese model and high-fat diet-fed mouse livers. These results propose that targeting the Drp1-filamin interaction become a new strategy for the prevention or treatment of fatty liver disease.