Aerobic training with moderate or high doses of vitamin D improve liver enzymes, LXRα and PGC-1α levels in rats with T2DM.

Dysregulation of key transcription factors involved in hepatic energy metabolism, such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and liver X receptor alpha (LXRα), has been observed in T2DM. The present study aims to investigate the effects of aerobic training and vitamin D supplementation on liver enzyme levels and the levels of PGC-1α and LXRα proteins in hepatocytes, in a rat model of T2DM. The study involved 56 male Wistar rats, divided into two groups: one was non-diabetic and acted as a control group (n = 8), and the other had induced diabetes (n = 48). The diabetic rats were then split into six subgroups: two groups received high or moderate doses of vitamin D and aerobic training (D + AT + HD and D + AT + MD); two groups received high or moderate doses of vitamin D alone (D + HD and D + MD); one group underwent aerobic training with vehicle (sesame oil; D + AT + oil), and one group was a diabetic control receiving only sesame oil (oil-receiving). The D + AT + HD and D + HD groups received 10,000 IU of vitamin D, while the D + AT + MD and D + MD groups received 5000 IU of vitamin D once a week by injection. The D + AT + oil group and the sham group received sesame oil. After eight weeks of treatment, body weight, BMI, food intake, serum insulin, glucose, 25-hydroxyvitamin D, ALT, AST, and visceral fat were measured. The levels of PGC-1α and LXRα proteins in the liver was assessed by western blotting. Statistical analysis was performed using the paired t-test, one-way analysis of variance (ANOVA), and the Tukey post hoc test at a significance level of P < 0.05. Body weight, food intake, and BMI decreased significantly in the D + AT + HD, D + AT + MD, D + AT + oil, D + HD, and D + MD groups with the highest reduction being observed in body weight and BMI in the D + AT + HD group. The D + AT + HD group exhibited the lowest levels of insulin, glucose, and HOMA-IR while the D + C group exhibited the highest levels among the diabetic groups. The D + AT + HD and D + AT + MD groups had lower levels of ALT and AST enzymes compared to the other groups with no significant difference between D + AT + HD and D + AT + MD. D + AT + HD (p = 0.001), D + AT + MD (p = 0.001), D + HD (p = 0.023), D + MD (p = 0.029), and D + AT + oil (p = 0.011) upregulated LXRα compared to D + C. Among these groups, D + AT + HD exhibited a more profound upregulation of LXRα than D + AT + MD, D + AT + oil, D + HD, and D + MD (p = 0.005; p = 0.002, p = 0.001, and p = 0.001, respectively). Similarly, D + AT + HD showed a more notable upregulation of PGC-1α compared to D + AT + oil, D + HD, and D + MD (p = 0.002; p = 0.001, and p = 0.001, respectively). Pearson correlation tests showed significant and negative correlations between serum 25-hydroxyvitamin levels and both visceral fat (r = - 0.365; p = 0.005) and HOMA-IR (r = - 0.118; p = 0.009); while positive and significant correlations between the liver-to-bodyweight ratio with both ALT and AST enzymes and also between QUICKI levels with LXRα (r = 0.578; p = 0.001) and PGC-1α (r = 0.628; p = 0.001). Combined administration of aerobic training and vitamin D supplementation potentially improves liver enzymes in type-2 diabetic rats that were simultaneous with upregulating the levels of PGC-1α and LXRα proteins in hepatocytes. These improvements were more significant when combining exercise with high-dose vitamin D supplementation. This study highlights the potential of this combination therapy as a new diabetes treatment strategy.

Therapeutic Potential of Aloe vera and Aloe vera-Conjugated Silver Nanoparticles on Mice Exposed to Hexavalent Chromium.

Hexavalent chromium (Cr (VI)) is a hazardous heavy metal that induces hepatotoxicity and nephrotoxicity. Thus, this study was planned to explore the ameliorating capacity of Aloe vera leaf gel extract (AV) and their conjugated silver nanoparticles (AVNP) against Cr (VI) induced hepatotoxicity and renal toxicity. The organ indices, level of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, malondialdehyde, total protein, and creatinine in blood serum were measured. The histopathological and micrometric analysis of the hepatic and renal tissue sections were studied. The hepatosomatic index was raised significantly (0.098 ± 0.13 g) in Cr treated group. The blood serum level of AST (484 ± 10.7 U/L), ALT (163 ± 5.5 U/L), ALP (336.7 ± 9.5 U/L), MDA (642.3 ± 28.3 U/L), and creatinine (4.0 ± 0.1 mg/dL) were increased significantly, whereas total protein level was declined (2.8 ± 0.3 g/dL) significantly in Cr exposed group. In the histopathological study, necrosis, disturbed hepatic cords, impaired glomeruli, and Bowman's capsule were noted. Micrometric data from the liver and kidney revealed a significant surge in the size of hepatocytes and their nuclei (1188.2 ± 467.7 µ2 and 456.5 ± 205.6 µ2) and CSA of glomeruli and Bowman's capsule (9051.8 ± 249.8 µ2 and 11,835.5 ± 336.7 µ2) in Cr (VI) exposed group, whereas the brush border (10.2 ± 4.0 µ) size declined significantly. The administration of AV and AVNP reduced the oxidative stress induced by Cr (VI).

Bisphenol A (BPA) exposure aggravates hepatic oxidative stress and inflammatory response under hypertensive milieu - Impact of low dose on hepatocytes and influence of MAPK and ER stress pathways.

Human exposure to the hazardous chemical, Bisphenol A (BPA), is almost ubiquitous. Due to the prevalence of hypertension (CVD risk factor) in the aged human population, it is necessary to explore its adverse effect in hypertensive subjects. The current study exposed the Nω-nitro-l-arginine methyl ester (L-NAME) induced hypertensive Wistar rats to human exposure relevant low dose of BPA (50 µg/kg) for 30 days period. The liver biochemical parameters, histopathology, immunohistochemistry, gene expression (RT-qPCR), trace elements (ICP-MS), primary rat hepatocytes cell culture and metabolomic (1H NMR) assessments were performed. Results illustrate that BPA exposure potentiates/aggravates hypertension induced tissue abnormalities (hepatic fibrosis), oxidative stress, ACE activity, malfunction of the antioxidant system, lipid abnormalities and inflammatory factor (TNF-α and IL-6) expression. Also, in cells, BPA increased ROS generation, mitochondrial dysfunction and lipid peroxidation without any impact on cytotoxicity and caspase 3 and 9 activation. Notably, BPA exposure modulate lipid metabolism (cholesterol and fatty acid) in primary hepatocytes. Finally, the influence of ERK1/2, p38MAPK, ER stress and oxidative stress during relatively high dose of BPA elicited cytotoxicity was observed. Therefore, a precise hazardous risk investigation of BPA exposure in hypertensive populations is highly recommended.

Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat.

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Reparative and Antioxidant Effects of New Analogues of Immunomodulator Thymogen in Experimental Model of Liver Damage.

We studied the reparative and antioxidant effects of Thymogen and its new structural analogues obtained by binding amino acid D-Ala to the N- or C-end of the peptide molecule in acute toxic hepatopathy. Intragastric administration of carbon tetrachloride for 5 days caused the development of fat degeneration of hepatocytes, a decrease in catalase activity, and an increase in malondialdehyde concentration. Administration of peptides suppressed oxidative peroxidation and stimulated reparative regeneration of hepatocytes; Thymogen analogues produced more pronounced hepatotropic and antioxidant effects than Thymogen. Inclusion of D-Ala enhanced the effect of Thymogen on the processes of regeneration in hepatocytes and the antioxidant effect under conditions of acute carbon tetrachloride hepatopathy. The highest efficiency was achieved when the amino acid was added to the C-end of the molecule.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 µm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

Branched-chain fatty acids affect the expression of fatty acid synthase and C-reactive protein genes in the hepatocyte cell line.

Fatty acids (FAs) are known to play an important role in human metabolism; however, still little is known about the functions of certain FA classes present in blood at relatively low concentrations. Examples of such compounds include branched-chain fatty acids (BCFAs). Recently, lowered BCFAs blood concentration was noticed in obese patients. An inverse correlation was found between serum concentrations of BCFAs and triglyceride levels, as well as C-reactive protein (CRP) concentration. Obesity is the most frequently observed component of metabolic syndrome and both disorders are accompanied by the dysregulation of FAs metabolism. However, not all of them are well understood. Our study is the first attempt at presenting the opposite effects of an iso-BCFA (14-methylpentadecanoic acid, 14-MPA) and an anteiso-BCFA (12-methyltetradecanoic acid, 12-MTA) on selected genes related to fatty acid synthesis and inflammation: FASN, SREBP1, CRP, and IL-6 in the HepG2 cell line. We observed lowered expression of FASN, SREBP1, CRP, and IL-6 in cells treated with 14-MPA in comparison with control cells. In contrast, supplementation with 12-MTA caused opposite effects: increased mRNA levels of FASN, CRP, and IL-6. 12-MTA did not influence SREBP1 expression. The results of our preliminary study may suggest potential benefits of the supplementation of iso-BCFAs in obese patients, for inflammation and hypertriglyceridemia prevention.

Efectos de fuentes de grasa sobre características del hígado y la morfometría intestinal en un modelo animal a temprana edad / Effects of fat sources on liver characteristics and intestinal morphometry in an early-life animal model

RESUMEN El objetivo fue determinar el efecto del consumo de tres fuentes de ácidos grasos sobre el peso relativo, caracterización macroscópica y microscópica del hígado, y la morfometría intestinal en un modelo animal a temprana edad. Se utilizaron 76 pollitos distribuidos al azar, que recibieron una de las dietas (T1: 97,0% de dieta basal (DB) + 3,0% de material inerte, T2: 97,0% de DB + 3,0% de manteca vegetal parcialmente hidrogenada, T3: 97,0% de DB + 3,0% de aceite de quinua, y T4: 97,0% de DB + 3,0% de aceite de pescado) hasta el séptimo día de vida; luego se extrajeron muestras para los análisis correspondientes. Se encontró que, los animales que consumieron aceite de quinua (T3) o aceite de pescado (T4) tuvieron resultados favorables asociados a un menor peso del hígado y, una mejor absorción de nutrientes a nivel intestinal debido a mayores valores en la relación longitud del vello y profundidad de cripta; en comparación a la manteca vegetal parcialmente hidrogenada (T2). En conclusión, el aceite de quinua constituye una opción saludable de consumo y fuente alternativa al aceite de pescado.

ABSTRACT We aimed to determine the effect of the consumption of three sources of fatty acids on the relative weight, macroscopic and microscopic characteristics of the liver, and intestinal morphometry in an early-life animal model. Seventy-six randomly distributed chicks received one of the diets (T1: 97.0% basal diet (BD) + 3.0% inert material, T2: 97.0% BD + 3.0% partially hydrogenated vegetable shortening, T3: 97.0% BD + 3.0% quinoa oil, and T4: 97.0% BD + 3.0% fish oil) until the seventh day of life; samples were then extracted in order to be analyzed. We found that the animals that consumed quinoa oil (T3) or fish oil (T4) had favorable results associated to lower liver weight and better absorption of nutrients at intestinal level due to higher values in the hair length and crypt depth ratio, in comparison to partially hydrogenated vegetable shortening (T2). In conclusion, quinoa oil constitutes a healthy option for consumption and an alternative source to fish oil.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects.

Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.

In Vitro/In Vivo Hepatoprotective and Antioxidant Effects of Defatted Extract and a Phenolic Fraction Obtained from Phlomis Tuberosa.

An in vitro/in vivo hepatotoxicity and hepatoprotection evaluation of a defatted extract and a phenolic fraction from Phlomis tuberosa, administered alone and in a carbon tetrachloride (CCl4)-induced metabolic bioactivation model, was performed. The extract and the phenolic fraction were analysed by high performance liquid chromatography (HPLC) to determine the total flavonoid content, to identify flavonoids and to quantify verbascoside. In addition, total polyphenolics in the samples were expressed as gallic acid equivalents. Applied alone, the extract and the fraction (5, 10 and 50 µg/mL) did not show a statistically significant hepatotoxic effect on isolated rat hepatocytes in vitro. In a CCl4-induced hepatotoxicity model, the samples exhibited a concentration-dependent, statistically significant hepatoprotective effect, which was most pronounced at 50 µg/mL for both. The phenolic fraction exhibited a more pronounced hepatoprotective effect compared to the extract. Data from the in vitro study on the effects of the extract were also confirmed in the in vivo experiment conducted in a CCl4-induced hepatotoxicity model in rats. A histopathological study showed that the animals treated with CCl4 and the extract had an unaltered histoarchitecture of the liver. The effects of the extract were the same as those of silymarin.

Heptadecanoic Acid Is Not a Key Mediator in the Prevention of Diet-Induced Hepatic Steatosis and Insulin Resistance in Mice.

Epidemiological studies found that the intake of dairy products is associated with an increased amount of circulating odd-chain fatty acids (OCFA, C15:0 and C17:0) in humans and further indicate that especially C17:0 is associated with a lower incidence of type 2 diabetes. However, causal relationships are not elucidated. To provide a mechanistic link, mice were fed high-fat (HF) diets supplemented with either milk fat or C17:0 for 20 weeks. Cultured primary mouse hepatocytes were used to distinguish differential effects mediated by C15:0 or C17:0. Despite an induction of OCFA after both dietary interventions, neither long-term milk fat intake nor C17:0 supplementation improved diet-induced hepatic lipid accumulation and insulin resistance in mice. HF feeding with milk fat actually deteriorates liver inflammation. Treatment of primary hepatocytes with C15:0 and C17:0 suppressed JAK2/STAT3 signaling, but only C15:0 enhanced insulin-stimulated phosphorylation of AKT. Overall, the data indicate that the intake of milk fat and C17:0 do not mediate health benefits, whereas C15:0 might be promising in further studies.

Circulating Human Metabolites Resulting from TOTUM-070 Absorption (a Plant-Based, Polyphenol-Rich Ingredient) Improve Lipid Metabolism in Human Hepatocytes: Lessons from an Original Ex Vivo Clinical Trial.

TOTUM-070 is a patented polyphenol-rich blend of five different plant extracts showing separately a latent effect on lipid metabolism and potential synergistic properties. In this study, we investigated the health benefit of such a formula. Using a preclinical model of high fat diet, TOTUM-070 (3 g/kg of body weight) limited the HFD-induced hyperlipemia with a reduction in triglyceride (-32% after 6 weeks; -20.3% after 12 weeks) and non-HDL cholesterol levels (-21% after 6 weeks; -38.4% after 12 weeks). To further investigate such a benefit and its underlying mechanisms in humans, we designed an ex vivo clinical approach to collect the circulating bioactives resulting from TOTUM-070 ingestion and to determine their biological activities on human hepatocytes. Human serum was obtained from healthy subjects before and after intake of TOTUM-070 (4995 mg). The presence of circulating metabolites was assessed by UPLC-MS/MS. Serum containing metabolites was further incubated with hepatocytes cultured in a lipotoxic environment (palmitate, 250 µM). RNA sequencing analyses show that lipid metabolism was one of the most impacted processes. Using histologic, proteomic, and enzymatic assays, the effects of human TOTUM-070 bioactives on hepatocyte metabolism were characterized by (1) the inhibition of lipid storage, including both (2) triglycerides (-41%, p < 0.001) and (3) cholesterol (-50%, p < 0.001) intracellular content, (4) a reduced de novo cholesterol synthesis (HMG-CoA reductase activity -44%, p < 0.001), and (5) a lowered fatty acid synthase protein level (p < 0.001). Altogether, these data support the beneficial impact of TOTUM-070 on lipid metabolism and provide new biochemical insights in human mechanisms occurring in liver cells.

Chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) leaf extracts regulate mitochondrial bioenergetics and fatty acid oxidation in C2C12 myotubes and primary hepatocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) is an important component of the regular diet and traditional medicine of indigenous communities in Mexico. Customarily, Chaya is consumed as a beverage made of macerated leaf, cooked, or prepared in teas or infusions to empirically treat obesity, diabetes, gastrointestinal disorders, and kidney stones. The beneficial effects of Chaya can be attributed to the presence of protein, dietary fiber, vitamins, and especially polyphenols, which regulate mitochondrial function. Therefore, polyphenols present in Chaya extracts could be used to develop novel strategies to prevent and treat metabolic alterations related to mitochondrial dysfunction in the muscle and liver of subjects with obesity, type 2 diabetes, and cardiovascular diseases. However, limited information is available concerning the effect of Chaya extracts on mitochondrial activity in those tissues. AIM OF THE STUDY: The aim of this study was to evaluate the antioxidant capacity of an aqueous extract (AE) or mixed (methanol/acetone/water) extract (ME) of Chaya leaf and their effect on C2C12 myotubes and primary hepatocyte mitochondrial bioenergetics and fatty acid oxidation (FAO). MATERIALS AND METHODS: Total polyphenol content and antioxidant activity were determined using the Folin-Ciocalteu method and the oxygen radical absorbance capacity assay, respectively. The effect of AE and ME from Chaya leaf on mitochondrial activity and FAO of C2C12 myotubes and primary hepatocytes was evaluated using an extracellular flux analyzer. RESULTS: The AE and ME from Chaya leaf exhibited antioxidant activity and a polyphenol content similar to nopal, another plant used in Mexican traditional medicine. AE significantly (p < 0.05) decreased the maximal respiration and spare respiratory capacity (SRC) of C2C12 cells, whereas ME had little effect on C2C12 mitochondrial function. Conversely, ME significantly (p < 0.05) decreased SRC in primary hepatocytes, whereas AE increased maximal respiration and SRC at low doses (5 and 10 µM). Moreover, low doses of Chaya AE significantly (p < 0.05) increased AMPK phosphorylation, acyl-coenzyme A oxidase protein abundance, and palmitate oxidation in primary hepatocytes. CONCLUSION: The AE of Chaya leaf increases mitochondrial function and FAO of primary hepatocytes, indicating its potential to treat hepatic mitochondrial dysfunction underlying metabolic diseases.

Nano-selenium protects grass carp hepatocytes against 4-tert-butylphenol-induced mitochondrial apoptosis and necroptosis via suppressing ROS-PARP1 axis.

4-tert-butylphenol (4-tBP) is a monomer widely used in the synthesis of industrial chemicals, and posed a high risk to aquatic animals. Our study focused on toxic phenotype and mechanism of detoxification in grass carp hepatocytes (L8824) after 4-tBP-treatment. In this experiment, L8824 displayed hallmark phenotypes of apoptosis and necroptosis after 4-tBP exposure, as evidenced by changes in cell morphology, increased rates of apoptosis and necrosis, the loss of MMP, the accumulation of ROS, and changes in associated factors (PARP1, JNK, Bid, Bcl-2, Bax, AIFM1, CytC, Caspase 9, APAF1, Caspase 3, TNF-α, TNFR1, RIPK1, RIPK3, and MLKL). Furthermore, we found that 4-tBP-induced apoptosis and necroptosis were reversed by pretreating with N-Acetylcysteine (a ROS scavenger) and 3-Aminobenzamide (a PARP1 inhibitor), indicating that 4-tBP induced the onset of mitochondrial apoptosis and necroptosis in L8824 via activating ROS-PARP1 axis. Nano-selenium (Nano-Se) is a novel form of Se with a noteworthy antioxidant capacity. Here, Nano-Se was found to have preventive, therapeutic, and resistance effects on 4-tBP-induced L8824 apoptosis and necroptosis. Nano-Se co-treatment with 4-tBP was an optimal way to alleviate 4-tBP-induced apoptosis and necroptosis. We demonstrated for the first time that Nano-Se protected L8824 against 4-tBP-induced mitochondrial apoptosis and necroptosis through ROS-PARP1 pathway. This study will provide a new theoretical basis for 4-tBP toxicology researches and aquatic animal protection.

In Vitro Hepatic Models to Assess Herb-Drug Interactions: Approaches and Challenges.

A newfound appreciation for the benefits of herbal treatments has emerged in recent decades. However, herbal medication production still needs to establish standardized protocols that adhere to strict guidelines for quality assurance and risk minimization. Although the therapeutic effects of herbal medicines are extensive, the risk of herb-drug interactions remains a serious concern, limiting their use. Therefore, a robust, well-established liver model that can fully represent the liver tissue is required to study potential herb-drug interactions to ensure herbal medicines' safe and effective use. In light of this, this mini review investigates the existing in vitro liver models applicable to detecting herbal medicines' toxicity and other pharmacological targets. This article analyzes the benefits and drawbacks of existing in vitro liver cell models. To maintain relevance and effectively express the offered research, a systematic strategy was employed to search for and include all discussed studies. In brief, from 1985 to December 2022, the phrases "liver models", "herb-drug interaction", "herbal medicine", "cytochrome P450", "drug transporters pharmacokinetics", and "pharmacodynamics" were combined to search the electronic databases PubMed, ScienceDirect, and the Cochrane Library.

Prediction of the mechanisms of action of Qutan Huoxue decoction in non-alcoholic steatohepatitis (NASH): a network pharmacology study and experimental validation.

CONTEXT: Qutan Huoxue decoction (QTHX) is used to treat non-alcoholic steatohepatitis (NASH) with good efficacy in the clinic. However, the mechanism is not clear yet. OBJECTIVE: This study investigates the mechanism of QTHX in the treatment of NASH. MATERIALS AND METHODS: Potential pathways of QTHX were predicted by network pharmacology. Fourty Sprague Dawley (SD) rats (half normal diet, half high-fat diet) were fed six to eight weeks, primary hepatocytes and Kupffer cells were extracted and co-cultured by the 0.4-micron trans well culture system. Then, the normal co-cultured cells were treated by normal serum, the NASH co-cultured cells were treated with various concentrations of QTHX-containing serum (0, 5, 7.5 or 10 µg/mL) for 24 h. The expression of targets were measured with Activity Fluorometric Assay, Western blot and PCR assay. RESULTS: Network pharmacology indicated that liver-protective effect of QTHX was associated with its anti-inflammation response, oxidative stress, and lipid receptor signalling. 10 µg/mL QTHX significantly reduced the inflammation response and lipid levels in primary hepatocytes (ALT: 46.43 ± 2.76 U/L, AST: 13.96 ± 1.08 U/L, TG: 0.25 ± 0.01 mmol/L, TC: 0.14 ± 0.05 mmol/L), comparing with 0 µg/mL NASH group (ALT: 148 ± 9.22 U/L, AST: 53.02 ± 2.30 U/L, TG: 0.74 ± 0.07 mmol/L, TC: 0.91 ± 0.07 mmol/L) (p < 0.01). Meanwhile, QTHX increased expression of SOCS1 and decreased expression of TLR4, Myd88, NF-κB. CONCLUSIONS: The study suggested that QTHX treats NASH in rats by activating the SCOS1/NF-κB/TLR4 pathway, suggesting QTHX could be further developed as a potential liver-protecting agent.

Transcriptomics of natural and synthetic vitamin D in human hepatocyte lipotoxicity.

Vitamin D (VD) has been used to prevent nonalcoholic fatty liver disease (NAFLD), a condition of lipotoxicity associated with a defective metabolism and function of this vitamin. Different forms of VD are available and can be used for this scope, but their effects on liver cell lipotoxicity remain unexplored. In this study we compared a natural formulation rich in VD2 (Shiitake Mushroom extract or SM-VD2) with a synthetic formulation containing pure VD3 (SV-VD3) and the bioactive metabolite 1,25(OH)2-D3. These were investigated in chemoprevention mode in human HepaRG liver cells supplemented with oleic and palmitic acid to induce lipotoxicity. All the different forms of VD showed similar efficacy in reducing the levels of lipotoxicity and the changes that lipotoxicity induced on the cellular transcriptome. However, the three forms of VD generated different gene fingerprints suggesting diverse, even if functionally convergent, cytoprotective mechanisms. Main differences were (1) the number of differentially expressed genes (SV-VD3 > 1,25[OH]2-D3 > SM-VD2), (2) their identity that demonstrated significant gene homology between SM-VD2 and 1,25(OH)2-D3, and (3) the number and type of biological functions identified by ingenuity pathway analysis as relevant to liver metabolism and cytoprotection annotations. Immunoblot confirmed a different response of VDR and other VDR-related proteins to natural and synthetic VD formulations, including FXR, PXR, PPARγ/PGC-1α, and CYP3A4 and CYP24A1. In conclusion, different responses of the cellular transcriptome drive the cytoprotective effect of natural and synthetic formulations of VD in the free fatty acid-induced lipotoxicity of human hepatocytes.

Nano-selenium Antagonizes Heat Stress-Induced Apoptosis of Rainbow Trout (Oncorhynchus mykiss) Hepatocytes by Activating the PI3K/AKT Pathway.

The cold-water fish rainbow trout (Oncorhynchus mykiss) shows poor resistance to heat, which is the main factor restricting their survival and yield. With the advancement of nanotechnology, nano-selenium (nano-Se) has emerged as a key nano-trace element, showing unique advantages, including high biological activity and low toxicity, for studying the response of animals to adverse environmental conditions. However, little is still known regarding the potential protective mechanisms of nano-Se against heat stress-induced cellular damage. Herein, we aimed to investigate the mechanism underlying the antagonistic effects of nano-Se on heat stress. Four groups were assessed: CG18 (0 µg/mL nano-Se, 18 °C), Se18 (5.0 µg/mL nano-Se, 18 °C), CG24 (0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture), and Se24 (5.0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture). We found that after heat treatment (CG24 group), T-AOC, GPx, and CAT activities in rainbow trout hepatocytes showed a decrease of 36%, 33%, and 19%, respectively, while ROS and MDA levels showed an increase of 67% and 93%, respectively (P < 0.05). Meanwhile, the mRNA levels of the apoptosis-related genes caspase3, caspase9, Cyt-c, Bax, and Bax/Bcl-2 in the CG24 group were 41%, 47%, 285%, 65%, and 151% higher than those in the CG18 group, respectively, while those of PI3K and AKT were 31% and 17% lower, respectively (P < 0.05). Besides, flow cytometry analysis showed an increase in the level of apoptotic cells after heat exposure. More importantly, we observed that nano-Se cotreatment (Se24 group) remarkably attenuated heat stress-induced effects (P < 0.05). We conclude that heat stress induces oxidative stress and apoptosis in rainbow trout hepatocytes. Nano-Se ameliorates heat stress-induced apoptosis by activating the PI3K/AKT pathway. Our results provide a new perspective to improve our understanding of the ability of nano-Se to confer heat stress resistance.

Deleterious liver-adipose crosstalk in obesity: Hydroethanolic extract of Lampaya medicinalis Phil. (Verbenaceae) counteracts fatty acid-induced fibrotic marker expression in human hepatocytes.

Elevated circulating fatty acids in obesity may induce hepatic steatosis, leading to liver inflammation, fibrosis and nonalcoholic fatty liver disease (NAFLD). On the other hand, impaired communication between hepatocytes and adipose tissue (AT) in obesity influences adipose lipolysis and fibrosis, negatively affecting metabolic function. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in Chilean folk medicine to treat inflammatory diseases. Hydroethanolic extract of lampaya (HEL) contains flavonoids that may explain its anti-inflammatory effect, but it is unknown whether HEL modulates fibrogenic processes in hepatocytes. We studied lipolysis and expression of fibrosis markers after exposure of visceral AT explants from subjects with obesity to HepG2-secreted factors. In addition, we evaluated the effect of HEL on palmitic acid (PA, C16:0) and oleic acid (OA; C18:1)-induced fibrotic marker expression in HepG2 hepatocytes. Results: Exposure to HepG2-secreted factors increased visceral AT lipolysis and expression of CTGF and collagen I. Exposure to OA/PA elevated collagen I, CTGF, fibronectin, α-smooth muscle actin, MMP-2 and MMP-9 expression in HepG2 cells, and these effects were prevented by HEL co-treatment. Conclusion: HEL effect counteracting OA/PA-induced fibrotic marker expression in HepG2 hepatocytes may represent a preventive approach against hepatic fibrosis and deleterious liver-adipose crosstalk in obesity.