Model Of Steatosis Research Articles

Hepatocyte-specific deletion of Ppar\u03b1 promotes NAFLD in the context of obesity

Peroxisome proliferator activated receptor α (PPARα) acts as a fatty acid sensor to orchestrate the transcription of genes coding for rate-limiting enzymes required for lipid oxidation in hepatocytes. Mice only lacking Pparα in hepatocytes spontaneously develop steatosis without obesity in aging. Steatosis can develop into non alcoholic steatohepatitis (NASH), which may progress to irreversible damage, such as fibrosis and hepatocarcinoma. While NASH appears as a major public health concern worldwide, it remains an unmet medical need. In the current study, we investigated the role of hepatocyte PPARα in a preclinical model of steatosis. For this, we used High Fat Diet (HFD) feeding as a model of obesity in C57BL/6 J male Wild-Type mice (WT), in whole-body Pparα- deficient mice (Pparα−/−) and in mice lacking Pparα only in hepatocytes (Pparαhep−/−). We provide evidence that Pparα deletion in hepatocytes promotes NAFLD and liver inflammation in mice fed a HFD. This enhanced NAFLD susceptibility occurs without development of glucose intolerance. Moreover, our data reveal that non-hepatocytic PPARα activity predominantly contributes to the metabolic response to HFD. Taken together, our data support hepatocyte PPARα as being essential to the prevention of NAFLD and that extra-hepatocyte PPARα activity contributes to whole-body lipid homeostasis.

Hepatic Choline Transport Is Inhibited During Fatty Acid-Induced Lipotoxicity and Obesity.

Choline is an essential nutrient and a critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine, while also contributing to the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the cytidine diphosphate–choline or the phosphatidylethanolamine N‐methyltransferase pathway. With the continuing global rise in the rates of obesity and nonalcoholic fatty liver disease, we sought to explore how excess fatty acids on primary hepatocytes and diet‐induced obesity affect choline uptake and metabolism. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared with control. The effects of palmitate treatment were independent of endoplasmic reticulum stress, which counterintuitively augmented choline transport and transporter expression. In a model of obesity‐induced hepatic steatosis, male mice fed a 60% high‐fat diet for 10 weeks had significantly diminished hepatic choline uptake compared with lean mice fed a control diet. Although the transcript and protein expression of various choline metabolic enzymes fluctuated slightly, we observed reduced protein expression of choline transporter‐like 1 (CTL1) in the liver of mice fed a high‐fat diet. Polysome profile analyses revealed that in livers of obese mice, the CTL1 transcript, despite being more abundant, was translated to a lesser extent compared with lean controls. Finally, human liver cells demonstrated a similar response to palmitate treatment. Conclusion: Our results suggest that the altered fatty acid milieu seen in obesity‐induced fatty liver disease progression may adversely affect choline metabolism, potentially through CTL1, but that compensatory mechanisms work to maintain phospholipid homeostasis.

In Vitro and In Vivo Nutraceutical Characterization of Two Chickpea Accessions: Differential Effects on Hepatic Lipid Over-Accumulation.

Dietary habits are crucially important to prevent the development of lifestyle-associated diseases. Diets supplemented with chickpeas have numerous benefits and are known to improve body fat composition. The present study was undertaken to characterize two genetically and phenotypically distinct accessions, MG_13 and PI358934, selected from a global chickpea collection. Rat hepatoma FaO cells treated with a mixture of free fatty acids (FFAs) (O/P) were used as an in vitro model of hepatic steatosis. In parallel, a high-fat diet (HFD) animal model was also established. In vitro and in vivo studies revealed that both chickpea accessions showed a significant antioxidant ability. However, only MG_13 reduced the lipid over-accumulation in steatotic FaO cells and in the liver of HFD fed mice. Moreover, mice fed with HFD + MG_13 displayed a lower level of glycemia and aspartate aminotransferase (AST) than HFD mice. Interestingly, exposure to MG_13 prevented the phosphorylation of the inflammatory nuclear factor kappa beta (NF-kB) which is upregulated during HFD and known to be linked to obesity. To conclude, the comparison of the two distinct chickpea accessions revealed a beneficial effect only for the MG_13. These findings highlight the importance of studies addressing the functional characterization of chickpea biodiversity and nutraceutical properties.

CCN1 promotes hepatic steatosis and inflammation in non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is characterized by increased uptake and accumulation of lipids in hepatocytes. Simple steatosis may progress to non-alcoholic steatohepatitis (NASH) with inflammation, hepatocellular injury and fibrosis. CCN1 is an important matrix protein that regulates cell death and promotes immune cell adhesion and may potentially control this process. The role of CCN1 in NASH remains unclear. We investigated the role of CCN1 in the pathogenesis of steatohepatitis. CCN1 upregulation was found to be closely related with steatosis in patients with NASH, obese mice and a FFA-treated hepatocyte model. Controlling the expression of CCN1 in murine NASH models demonstrated that CCN1 increased the severity of steatosis and inflammation. From the sequence results, we found that fatty acid metabolism genes were primarily involved in the MCD mice overexpressing CCN1 compared to the control. Then, the expression of fatty acid metabolism genes was determined using a custom-designed pathway-focused qPCR-based gene expression array. Expression analysis showed that CCN1 overexpression significantly upregulated the expression of fatty acid metabolism-associated genes. In vitro analysis revealed that CCN1 increased the intracellular TG content, the pro-inflammatory cytokines and the expression level of apoptosis-associated proteins in a steatosis model using murine primary hepatocytes. We identified CCN1 as an important positive regulator in NASH.

Effect of knockdown of O-Glcnac transferase on hepatocyte fat synthesis

Objective: To investigate the effect of knockdown of O-GlcNAc transferase (OGT) on hepatocyte fat synthesis. Methods: Liver cell line L02 were used to established the model of hepatic steatosis. The levels of OGT and O-GlcNAc protein were detected by Western blot. The OGT knockdown cell line of L02 cells was established, and its lipid formation ability was detected after induction of oleic acid (OA). Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect mRNA and protein expression of enzymes related to fat synthesis. An independent sample t test was used. Results: Western blot showed that the expression of OGT and O-GlcNAc was increased in L02 cells after adipogenesis (P < 0.05). After shOGT lentivirus infects L02 cells, OGT mRNA levels were down-regulated (P < 0.01). Oil red O staining showed that the lipid in L02 shOGT cells decreased, qRT-PCR showed that the mRNA expressions of fat synthase (ACC1), (FASN) and (SCD1) were decreased, the difference was statistically significant (P < 0.05), protein Expression is consistent with mRNA expression. Conclusion: Knockdown of OGT can inhibit hepatocyte fat synthesis by reducing O-GlcNAc levels.

Use of an in vitro model of hepatic steatosis for studying the anti-oxidant and antisteatotic effects of fucoidan polysaccharides

Non Alcoholic Fatty Liver Disease (NAFLD) is characterised by fat accumulation in hepatocytes in the form of triacyglycerols (TAGs) within cytosolic lipid droplets. Fucoidans (FUs) are biologically active polysaccharides usually isolated from brown marine algae, but recently identified also in terrestrial plants. In this study, we aimed to investigate the anti-oxidant and anti-steatotic effects of FUs purified from C. compressa, F. hermonis, and E. globulus. To this aim, we used a validated NAFLD in vitro model consisting of rat hepatoma FaO cells exposed to an oleate/palmitate mixture. Such a model is suitable for rapid investigation of direct effects of natural and artificial compounds, together with satisfying the strategy of 3Rs for laboratory use of animals. Our results indicated that all FUs display anti-oxidant and anti-steatotic activities. Steatotic FaO cells may be employed to further study the biological effects of FUs.

Development of Prediction Models for Drug-Induced Cholestasis, Cirrhosis, Hepatitis, and Steatosis Based on Drug and Drug Metabolite Structures.

Drug-induced liver injury (DILI) is one of the major reasons for termination of drug development. Due to the importance of predicting DILI in early phases of drug development, diverse in silico models have been developed to filter out DILI-causing candidates before clinical study. However, no computational models have achieved sufficient prediction power for screening DILI in early phases because 1) drugs often cause liver injury through reactive metabolites, 2) different clinical outcomes of DILI have different mechanisms, and 3) the DILI label on drugs is not clearly defined. In this study, we developed binary classification models to predict drug-induced cholestasis, cirrhosis, hepatitis, and steatosis based on the structure of drugs and their metabolites. DILI-positive data was obtained from post-market reports of drugs and DILI-negative data from DILIrank, a database curated by the Food and Drug Administration (FDA). Support vector machine (SVM) and random forest (RF) were used in developing models with nine fingerprints and one 2D molecular descriptor calculated from drug (152 DILI-positives and 102 DILI-negatives) and drug metabolite (192 DILI-positives and 126 DILI-negatives) structures. Models were developed according to Organisation for Economic Co-operation and Development (OECD) guidelines for quantitative structure-activity relationship (QSAR) validation. Internal and external validation was performed with a randomization test in order to thoroughly examine model predictability and avoid random correlation between structural features and adverse outcomes. The applicability domain was defined with a leverage method for reliable prediction of new chemicals. The best models for each liver disease were selected based on external validation results from drugs (cholestasis: 70%, cirrhosis: 90%, hepatitis: 83%, and steatosis: 85%) and drug metabolites (cholestasis: 86%, cirrhosis: 88%, hepatitis: 86%, and steatosis: 83%) with applicability domain analysis. Compiled data sets were further exploited to derive privileged substructures that were more frequent in DILI-positive sets compared to DILI-negative sets and in drug metabolite structures compared to drug structures with a Morgan fingerprint level 2.

In Steatotic Cells, ATP-Citrate Lyase mRNA Is Efficiently Translated through a Cap-Independent Mechanism, Contributing to the Stimulation of De Novo Lipogenesis.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease in which excessive amount of lipids is accumulated as droplets in hepatocytes. Recently, cumulative evidences suggested that a sustained de novo lipogenesis can play an important role in NAFLD. Dysregulated expression of lipogenic genes, including ATP-citrate lyase (ACLY), has been found in liver diseases associated with lipid accumulation. ACLY is a ubiquitous cytosolic enzyme positioned at the intersection of nutrients catabolism and cholesterol and fatty acid biosyntheses. In the present study, the molecular mechanism of ACLY expression in a cell model of steatosis has been reported. We identified an internal ribosome entry site (IRES) in the 5′ untranslated region of the ACLY mRNA, that can support an efficient mRNA translation through a Cap-independent mechanism. In steatotic HepG2 cells, ACLY expression was up-regulated through IRES-mediated translation. Since it has been demonstrated that lipid accumulation in cells induces endoplasmic reticulum (ER) stress, the involvement of this cellular pathway in the translational regulation of ACLY has been also evaluated. Our results showed that ACLY expression was increased in ER-stressed cells, through IRES-mediated translation of ACLY mRNA. A potential role of the Cap-independent translation of ACLY in NAFLD has been discussed.

Patchouli Oil Attenuates High Fat Diet-induced Non-alcoholic Hepatic Steatosis.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. Nevertheless, no first-line therapy exists. Hepatic steatosis is the earliest stage of NAFLD, which is characterized by an accumulation of hepatic lipids. Patchouli oil (PO), which is isolated from the well-known Chinese herb named Pogostemon cablin (Blanco) Benth. (Lamiaceae), inhibits hepatic lipid accumulation effectively. However, its potential ability for the treatment of NAFLD had not been reported before. Thus, the objective of this study was to investigate the effectiveness of PO against hepatic steatosis and its underlying mechanisms. We used a high fat diet (HFD)-induced hepatic steatosis model of rats to estimate the effect of PO against NAFLD. Hematoxylin-eosin and oil red O staining were used to analyze the hepatic histopathological changes. ELISA, RT-qPCR, and Western blotting analysis were applied to evaluate the parameters for hepatic steatosis. Our results showed that PO significantly attenuated the lipid profiles and the serum enzymes, evidenced by quantitative and histopathological analyses. It also markedly down-regulated the expression of sterol regulatory element-binding protein 1 (SREPB-1c) with its downstream factors in de novo lipogenesis. And, likewise, in lipid export by very low-density lipoproteins (VLDL), related molecules were dramatically improved. Furthermore, PO observably normalized the aberrant peroxisome proliferator-activated receptor α (PPAR-α) signal in fatty acids oxidation. In conclusion, PO exerted a preventing effect against HFD-induced steatosis and might be due to decrease de novo lipogenesis, promote export of lipids, as well as owing to improve fatty acids oxidation.

Chitooligosaccharides Modulate Glucose-Lipid Metabolism by Suppressing SMYD3 Pathways and Regulating Gut Microflora.

Chitooligosaccharides (COS) have a variety of biological activities due to their positively charged amino groups. Studies have shown that COS have antidiabetic effects, but their molecular mechanism has not been fully elucidated. The present study confirmed that COS can reduce hyperglycemia and hyperlipidemia, prevent obesity, and enhance histological changes in the livers of mice with type 2 diabetes mellitus (T2DM). Additionally, treatment with COS can modulate the composition of the gut microbiota in the colon by altering the abundance of Firmicutes, Bacteroidetes, and Proteobacteria. Furthermore, in T2DM mice, treatment with COS can upregulate the cholesterol-degrading enzymes cholesterol 7-alpha-hydroxylase (CYP7A1) and incretin glucagon-like peptide 1 (GLP-1) while specifically inhibiting the transcription and expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the key enzyme in cholesterol synthesis. Furthermore, using an oleic acid-induced hepatocyte steatosis model, we found that HMGCR can be directly transactivated by SET and MYND domain containing 3 (SMYD3), a transcriptional regulator, via 5′-CCCTCC-3′ element in the promoter. Overexpression of SMYD3 can suppress the inhibitory effect of COS on HMGCR, and COS might regulate HMGCR by inhibiting SMYD3, thereby exerting hypolipidemic functions. To the best of our knowledge, this study is the first to illustrate that COS mediate glucose and lipid metabolism disorders by regulating gut microbiota and SMYD3-mediated signaling pathways.

The impact of uric acid treatment on mitochlondria morphology and function in a steatosis model of L-02 cells

Background and objectives: Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic diseases worldwide. Oxidative stress (OS) is a major contributor toward NAFLD development, while mitochondria play a central role in OS. Our previous study has shown that uric acid (UA), as a dual function metabolite, could alleviate OS. This study examined the impact of UA on mitochondria morphology and function in a model of steatosis using L-02 cells to explore the pathogenesis of NAFLD. Methods: The L‑02 hepatocyte cell line was used to develop a steatosis cell model via 0.3 mM oleic acid (OA) over 24 h, subsequently treated with uric acid (UA) dose of 5, 10, and 20 mg/dL for 24, 48, and 72 h, respectively. The fluorescence intensity of reactive oxygen species (ROS), apoptosis rate, and activity of Succinate dehydrogenase(SDH), cytochrome oxidas(CCO), and adenosine triphosphate(ATP) synthase in electron transport chain (ETC), as well as the content of ATP and 8-OH-dG were examined; ultrastructure was observed under an electron microscope. Results: Treatment with UA at a concentration of 5 and 10 mg/dL decreased the rate of ROS production, apoptosis, and 8-OH-dG concentration, while supporting ATP recovery, and SDH activity in the steatosis model cell. It also promoted lipid droplet metabolism; however, the recovery of mitochondria morphology was not obvious. Conclusions: Treatment with UA dose of 5 and 10 mg/dL could protect mitochondria from OS damage. Future research requires a more stable and effective model. keywords : electron transport chain, oxidative stress, nonalcoholic fatty liver disease

Novel defatting strategies reduce lipid accumulation in primary human culture models of liver steatosis.

ABSTRACTNormothermic perfusion provides a means to rescue steatotic liver grafts, including by pharmacological defatting. In this study, we tested the potential of new drug combinations to trigger defatting in three human culture models, primary hepatocytes with induced steatosis, primary hepatocytes isolated from steatotic liver, and precision-cut liver slices (PCLS) of steatotic liver. Forskolin, L-carnitine and a PPARα agonist were all combined with rapamycin, an immunosuppressant that induces autophagy, in a D-FAT cocktail. D-FAT was tested alone or in combination with necrosulfonamide, an inhibitor of mixed lineage kinase domain like pseudokinase involved in necroptosis. Within 24 h, in all three models, D-FAT induced a decrease in triglyceride content by 30%, attributable to an upregulation of genes involved in free fatty acid β-oxidation and autophagy, and a downregulation of those involved in lipogenesis. Defatting was accompanied by a decrease in endoplasmic reticulum stress and in the production of reactive oxygen species. The addition of necrosulfonamide increased the efficacy of defatting by 8%-12% in PCLS, with a trend towards increased autophagy. In conclusion, culture models, notably PCLS, are insightful to design strategies for liver graft rescue. Defatting can be rapidly achieved by combinations of drugs targeting mitochondrial oxidative metabolism, macro-autophagy and lipogenesis.

DHA Protects Against Hepatic Steatosis by Activating Sirt1 in a High Fat Diet-Induced Nonalcoholic Fatty Liver Disease Mouse Model.

AimDocosahexaenoic acid (DHA; C22; n-3) shows beneficial effects on Non-alcoholic fatty liver disease (NAFLD). Deacetylase Sirtuin1 (Sirt1) was reported to increase energy metabolism and decrease lipogenesis. Here, we investigated whether DHA plays a role in protecting against hepatic steatosis via Sirt1.Main MethodsBoth in vivo and in vitro hepatic steatosis models were used: diet-induced obesity (DIO) model (middle-aged C57BL/6 mice fed a high-fat diet (HFD)) and palmitic acid (PA)-induced lipid accumulation cell model (HepG2 cells).Key FindingsIn DIO mice, treatment with DHA (gavage supplementation) for 8 weeks not only inhibited the lipid accumulation, but also increased fatty acids (FA) oxidation and induced triglyceride export in liver. These changes were accompanied by attenuation of inflammation. Moreover, DHA reversed the HFD-induced reduction of Sirt1 in liver. Interestingly, the beneficial effects of DHA were reversed by lentivirus-mediated Sirt1 knockdown, accompanied with increased expression of markers of lipogenesis, inflammation and reduced FA oxidation. In HepG2 cells, DHA prevented the accumulation of PA-induced lipid droplets, the decrease of FA oxidation and the reduction of Sirt1 level. Inhibition of Sirt1 by sirtinol partially reversed the beneficial effects of DHA on PA-treated cells.SignificanceDHA alleviated hepatic steatosis and reduced inflammation of liver in obese middle-aged mice by mechanisms involving Sirt1 activation.

Standardized rice bran extract improves hepatic steatosis in HepG2 cells and ovariectomized rats.

BACKGROUD/OBJECTIVESHepatic steatosis is the most common liver disorder, particularly in postmenopausal women. This study investigated the protective effects of standardized rice bran extract (RBS) on ovariectomized (OVX)-induced hepatic steatosis in rats.MATERIALS/METHODSHepG2 cells were incubated with 200 µM oleic acid to induce lipid accumulation with or without RBS and γ-oryzanol. OVX rats were separated into three groups and fed a normal diet (ND) or the ND containing 17β-estradiol (E2; 10 µg/kg) and RBS (500 mg/kg) for 16 weeks.RESULTSRBS supplementation improved serum triglyceride and free fatty acid levels in OVX rats. Histological analysis showed that RBS significantly attenuated hepatic fat accumulation and decreased hepatic lipid, total cholesterol, and triglyceride levels. Additionally, RBS suppressed the estrogen deficiency-induced upregulation of lipogenic genes, such as sterol regulatory element-binding protein 1 (SREBP1), acetyl-CoA carboxylase 1, fatty acid synthase, glycerol-3-phosphate acyltransferase, and stearoyl-CoA desaturase 1.CONCLUSIONSRBS and γ-oryzanol effectively reduced lipid accumulation in a HepG2 cell hepatic steatosis model. RBS improves OVX-induced hepatic steatosis by regulating the SREBP1-mediated activation of lipogenic genes, suggesting the benefits of RBS in preventing fatty liver in postmenopausal women.

Sarcopoterium spinosum Inhibited the Development of Non-Alcoholic Steatosis and Steatohepatitis in Mice.

Non-alcoholic fatty liver disease (NAFLD) is a comorbidity of obesity, which gradually develops from hepatic steatosis into steatohepatitis (NASH) and eventually even into fibrosis or hepatic carcinoma. To date, there has been no specific and effective treatment for NAFLD. Sarcopoterium spinosum extract (SSE) was found to improve insulin sensitivity. Recognizing the intimate link between insulin resistance and NAFLD, the aim of this study was to investigate the effectivity of SSE in the prevention and management of NAFLD at various severities. SSE was given to high-fat diet (HFD)-fed mice (steatosis model) or to mice given a Western diet (WD) in the short or long term (NASH prevention or treatment, respectively). SSE reduced body weight accumulation, improved glucose tolerance and insulin sensitivity and prevented the development of hepatic steatosis. SSE also blocked the progression of liver disease toward NASH in a dose-dependent manner. The expression of genes involved in lipid metabolism, inflammation, and antioxidant machinery was regulated by SSE in both models of steatosis and NASH development. However, SSE failed to reverse the hepatic damage in the advanced model of NASH. In summary, SSE might be considered as a botanical supplement for the prevention and treatment of hepatic steatosis, and for slowing the deterioration toward NASH.

Resveratrol Treatment Enhances the Cellular Response to Leptin by Increasing OBRb Content in Palmitate-Induced Steatotic HepG2 Cells.

The interaction of leptin with its hepatic longest receptor (OBRb) promotes the phosphorylation of signal transducer and activator of transcription-3 (STAT3), protecting the liver from lipid accumulation. However, leptin signalling is disrupted in hepatic steatosis, causing leptin resistance. One promising strategy to combat this problem is the use of bioactive compounds such as polyphenols. Since resveratrol (RSV) is a modulator of lipid homeostasis in the liver, we investigated whether treatment with different doses of RSV restores appropriate leptin action and fat accumulation in palmitate-induced steatotic human hepatoma (HepG2) cells. Both RSV metabolism and the expression of molecules implicated in leptin signalling were analysed. RSV at a 10 μM concentration was entirely metabolized to resveratrol-3-sulfate after 24 and counteracted leptin resistance by increasing the protein levels of OBRb. In addition, RSV downregulated the expression of lipogenic genes including fatty acid synthase (Fas) and stearoyl-CoA desaturase-1 (Scd1) without any significant change in Sirtuin-1 (SIRT1) enzymatic activity. These results demonstrate that RSV restored leptin sensitivity in a cellular model of hepatic steatosis in a SIRT1-independent manner.

Fatty Acids and Effects on In Vitro and In Vivo Models of Liver Steatosis.

Fatty liver, or steatosis, is a condition of excess accumulation of lipids, mainly under form of triglycerides (TG), in the liver, and it is the hallmark of non-alcoholic fatty liver disease (NAFLD). NAFLD is the most common liver disorder world-wide and it has frequently been associated with obesity, hyperlipidemia and insulin resistance. Free fatty acids (FA) are the major mediators of hepatic steatosis; patients with NAFLD have elevated levels of circulating FA that correlate with disease severity. Steatosis is a reversible condition that can be resolved with changed behaviors, or that can progress towards more severe liver damages such as steatohepatitis (NASH), fibrosis and cirrhosis. In NAFLD, FA of exogenous or endogenous origin accumulate in the hepatocytes and trigger liver damages. Excess TG are stored in cytosolic lipid droplets (LDs) that are dynamic organelles acting as hubs for lipid metabolism. In the first part of this review, we briefly reassumed the main classes of FA and their chemical classification as a function of the presence and number of double bonds, their metabolic pathways and effects on human health. Then, we summarized the main genetic and diet-induced animal models of NAFLD, as well as the cellular models of NAFLD. In recent years, both the diet-induced animal models of NAFLD as well as the cellular models of NAFLD have found ever more application to investigate the mechanisms involved in NAFLD, and we referred to their advantages and disadvantages.

Mechanism of Calculus Bovis Sativus in inhibiting hepatocyte lipid deposition based on serum pharmacology

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.

Beyond the Scavenging of Reactive Oxygen Species (ROS): Direct Effect of Cerium Oxide Nanoparticles in Reducing Fatty Acids Content in an In Vitro Model of Hepatocellular Steatosis

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic accumulation of lipids. Antisteatotic effects of cerium oxide nanoparticles (CeO2NPs) have recently been shown in animal models of liver disease. However, it is unclear whether the activity of CeO2NPs is related solely to the decrease in oxidative stress or, in addition, they directly decrease liver fatty acid accumulation. To address this question, in this work, we used an in vitro model of hepatocellular steatosis, exposing HepG2 cells to oleic and palmitic acid. Cell uptake of CeO2NPs and their effect on oxidative stress and viability of hepatic cells cultured with H2O2 were also evaluated. Results show that CeO2NPs were uptaken by HepG2 cells and reduced oxidative stress and improved cell viability. Treatment with oleic and palmitic acid increased lipogenesis and the content of different fatty acids. CeO2NPs reduced palmitic and stearic acid and most fatty acids consisting of more than 18 carbon atoms. These effects were associated with significant changes in elongase and desaturase activity. In conclusion, CeO2NPs directly protected HepG2 cells from cell injury in oxidative stress conditions and reduced fatty acid content in steatotic conditions by inducing specific changes in fatty acid metabolism, thus showing potential in the treatment of NAFLD.

Targeting CYP4A attenuates hepatic steatosis in a novel multicellular organotypic liver model

BackgroundNon-alcoholic fatty liver disease (NAFLD) begins as simple hepatic steatosis, but further progress to chronic liver diseases results in severe liver damage and hepatic failure. However, therapeutic options are scarce due to the lack of reliable human in vitro liver models for understanding disease progression mechanisms and developing therapies.ResultsWe describe here a novel method for generating 3D hepatic spheroids using HepaRG cells, vascular endothelial cells, and mesenchymal stem cells cultured on a thick layer of soft matrix in a narrow conical tube; this method improved self-organization efficiency and functional competence. We further developed a 3D hepatic steatosis model with excess glucose and palmitate, accurately recapitulating steatosis phenotypes such as neutral lipid accumulation, enhanced expression of lipogenesis and gluconeogenesis markers, increased intracellular triglyceride content, and reduced glucose uptake. The expression and activity of cytochrome P450 4A (CYP4A), a hepatic glucose and lipid homeostasis enzyme, that is highly expressed in liver tissues from NAFLD patients, was induced in our in vitro steatosis model, and inhibiting CYP4A with the selective inhibitor HET0016 or a specific siRNA ameliorated steatosis-related pathology through reduced ER stress and improved insulin signaling.ConclusionsWe provide here a novel 3D human cell-based hepatic model that can be easily generated and reliably simulate hepatic steatosis pathology. We have experimentally validated its potential for target validation and drug evaluation by focusing on CYP4A, which may serve as a translational platform for drug development.