AML12 Cells Research Articles

Selegiline ameliorated dyslipidemia and hepatic steatosis in high-fat diet mice.

Certain monoamine oxidase (MAO) inhibitors exhibit beneficial effects, such as reducing adiposity and metabolic disorders; however, their effects on hepatic lipid metabolism have not been revealed. This study aimed to investigate the effects of a selective MAO-B inhibitor, selegiline, on dyslipidemia and hepatic steatosis in mice induced by a high-fat diet (HFD). Administration of selegiline (0.6mg/kg body weight) by intraperitoneal injection was found to reduce HFD-induced body weight gain and increases in liver and adiposity coefficients, blood lipids and fatty acid levels. Furthermore, selegiline dramatically reduced the total triglyceride (TG) and cholesterol (TC) levels and lipid accumulation in the livers of HFD-fed mice and palmitic acid (PA)-treated AML-12 hepatocytes. In vivo and in vitro results indicated that selegiline protects against HFD- and PA-induced hepatic inflammation by reducing the expression of proinflammatory cytokines, namely IL-6, TNF-α, IL-1β, and IL-1α. Additionally, selegiline exhibited antioxidative effects on HFD and PA exposure in mouse liver and AML-12 cells by decreasing the levels of reactive oxygen species (ROS) and malonaldehyde (MDA) and increasing superoxide dismutase (SOD) activity. Further study showed that selegiline administration mitigated the expression of Srebf-1, Fasn, and Acaca and downregulated the expression of Cpt-1 and Pparα in HFD-fed mouse livers and PA-treated AML-12 cells. In conclusion, our findings suggest that selegiline exerts protective effects against HFD-induced dyslipidemia and hepatic steatosis, which may be related to an improved inflammatory response, oxidative stress, and hepatic lipid metabolism.

Rhynchophylline relieves nonalcoholic fatty liver disease by activating lipase and increasing energy metabolism.

Hepatic fat metabolism may be altered in the context of overnutrition and obesity, often resulting in the accumulation of triglycerides in hepatocytes and leading to nonalcoholic fatty liver disease (NAFLD). Natural plant alkaloids have demonstrated great potential for the prevention and treatment of NAFLD. However, the role of rhynchophylline (RHY) in lipid metabolism is not clear. We explored the role of RHY in lipid metabolism in cells treated with oleic and palmitic acids to mimic high-fat diet (HFD) conditions. RHY attenuated oleic and palmitic acid-induced increases in triglyceride accumulation in HepG2, AML12, and LMH cells. RHY also increased energy metabolism and reduced oxidative stress. We further investigated the effect of RHY on hepatic lipid metabolism in mice fed an HFD including 40mg/kg RHY. RHY alleviated hepatic steatosis, reduced fat deposition, promoted energy metabolism, and improved glucose metabolism. We investigated the mechanism responsible for this activity by docking with key proteins of lipid metabolism disorders using Discovery Studio software, which showed that RHY interacted well with lipases. Finally, we found that adding RHY promoted lipase activity and lipolysis. In conclusion, RHY ameliorated HFD-induced NAFLD and its complications by increasing lipase activity.

AMP-activated protein kinase-farnesoid X receptorpathway contributes to oleanolic acid-induced liver injury.

Natural pentacyclic triterpenoid oleanolic acid (OA) is used as an over-the-counter drug for acute and chronic hepatitis. However, clinical use of OA-containing herbal medicines has been reported to cause cholestasis, and the specific mechanism is unknown. The purpose of this study was to explore how OA causes cholestatic liver injury via the AMP-activated protein kinase (AMPK)-farnesoid X receptor (FXR) pathway. In animal experiments, it was found that OA treatment activated AMPK and decreased FXR and bile acid efflux transport proteins expression. When intervened with the specific inhibitor Compound C (CC), it was observed that AMPK activation was inhibited, the reduction of FXR and bile acid efflux transport protein expression was effectively alleviated, serum biochemical indicators were significantly reduced, and liver pathological damage brought about by OA was effectively ameliorated. In addition, OA was found to downregulate the expression of FXR and bile acid efflux transport proteins by activating the ERK1/2-LKB1-AMPK pathway in cellular experiments. The ERK1/2 inhibitor U0126 was used to pretreat primary hepatocytes, and this drastically reduced the phosphorylation levels of LKB1 and AMPK. The inhibition effects of OA on FXR and bile acid efflux transport proteins were also effectively alleviated after pretreatment with CC. In addition, OA-induced downregulation of FXR gene and protein expression levels was significantly prevented after silencing AMPKα1 expression in AML12 cells. Our study demonstrated that OA inhibited FXR and bile acid efflux transporters through the activation of AMPK, thus leading to cholestatic liver injury.

Effect of L-Dopa on Carboplatin Induced Hepatotoxicity

Carboplatin is an alkylating antineoplastic drug. Although carboplatin induces apoptosis in rapidly dividing cells like cisplatin, it has fewer side effects. The use of platinum compounds as antineoplastic agents at high doses causes hepatotoxicity, which is important in dose regulation. Levodopa is used as a precursor for dopamine synthesis in the brain in the treatment of Parkinson's disease. Dopamine synthesized in the liver from levodopa cannot cross the blood-brain barrier. There are studies suggesting that dopamine may have a protective effect on hepatocytes. In our study, we aimed to evaluate the cytoprotective effect of levodopa in cell damage induced by carboplatin in the liver cell line. Cell viability was evaluated by MTT test by exposing AML-12 hepatocyte cells to the combination of Carboplatin, L-DOPA and Carboplatin + L-DOPA for 24 hours (0, 3.13, 6.25, 12.5, 25, 50 ,100 µM) has been left. The LD50 value of carboplatin was determined as 62.65 µM. The LD50 values of the combination of L-DOPA and carboplatin + L-DOPA were higher in the tested dose range. The cytotoxic effect of carboplatin on the AML-12 cell line was determined by the increasing LD50 value, which was statistically significantly reduced when combined with levodopa. Further studies are needed to explain the mechanism of action.

FGF1 ameliorates obesity-associated hepatic steatosis by reversing IGFBP2 hypermethylation.

Obesity is a major contributing factor for metabolic-associated fatty liver disease (MAFLD). Fibroblast growth factor (FGF) 1 is the first paracrine FGF family member identified to exhibit promising metabolic regulatory properties capable of conferring glucose-lowering and insulin-sensitizing effect. This study explores the role and molecular underpinnings of FGF1 in obesity-associated hepatic steatosis. In a mouse high-fat diet (HFD)-induced MAFLD model, chronic treatment with recombinant FGF1(rFGF1) was found to effectively reduce the severity of insulin resistance, hyperlipidemia, and inflammation. FGF1 treatment decreased lipid accumulation in the mouse liver and palmitic acid-treated AML12 cells. These effects were associated with decreased mature form SREBF1 expression and its target genes FASN and SCD1. Interestingly, we uncovered that rFGF1 significantly induced IGFBP2 expression at both mRNA and protein levels in HFD-fed mouse livers and cultured hepatocytes treated with palmitic acid. Adeno-associated virus-mediated IGFBP2 suppression significantly diminished the therapeutic benefit of rFGF1 on MAFLD-associated phenotypes, indicating that IGFBP2 plays a crucial role in the FGF1-mediated reduction of hepatic steatosis. Further analysis revealed that rFGF1 treatment reduces the recruitment of DNA methyltransferase 3 alpha to the IGFBP2 genomic locus, leading to decreased IGFBP2 gene methylation and increased mRNA and protein expression. Collectively, our findings reveal FGF1 modulation of lipid metabolism via epigenetic regulation of IGFBP2 expression, and unravel the therapeutic potential of the FGF1-IGFBP2 axis in metabolic diseases associated with obesity.

Supression Thioredoxin reductase 3 exacerbates the progression of liver cirrhosis via activation of ferroptosis pathway

AimsIn the past decades, Txnrd3 as selenoprotein is considered to be highly expressed in testis and participate in sperm mature; however its role in liver diseases needs further study. Iron is essential for humans and animals, while its overload could damage to multiple organs. However, role of Txnrd3 and iron in cirrhosis is still unclear. Materials and methodsForty 8-week-old wild-type and forty Txnrd3−/− mice were selected to build liver cirrhosis model using Thiacetamide solution, deposition of iron in liver was observed via Prussian blue staining. Txnrd3 overexpression/knockdown model in vitro was constructed based on cell transfection techniques in AML12 cells, expression abundance of ferroptosis pathway genes within cells and tissues were determined by qRT-PCR and Western Blot. Key findingsResults showed that Txnrd3−/− mice developed more pronounced liver damage, accompanied by reduced GPX4 expression and iron deposition. A significant decrease in the expression abundance of GPX4 was also detected in Txnrd3 knock-down AML12 cells. In summary, Txnrd3 knockdown could result in iron overload and ferroptosis pathway activation within liver tissues and hepatocytes, ultimately lead to the occurrence of liver injury and cirrhosis. SignificanceThese results will provide biological markers for early diagnosis during cirrhosis and lay a theoretical basis for clinical therapy.

Di(2‐ethylhexyl) phthalate and microplastics cause necroptosis and apoptosis in hepatocytes of mice by inducing oxidative stress

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer and an endocrine disruptor. Microplastics (MPs) are pathogenic small plastic particles and abundant in the aqueous environment. The problem of residual hazards of plastic products is worthy of study, especially the joint exposure of a variety of plastic-related products to the toxic effect. We used 200 mg/kg DEHP and 10mg/L MPs to establish exposure model in vivo and 2 mM DEHP and 200 μg/L MPs to establish AML12 cell exposure model in vitro. In vivo study results showed that compared with the control group (NC) group, DEHP and MPs significantly increased the contents of malondialdehyde and hydrogen peroxide, and significantly decreased the contents of glutathione and the activity of superoxide dismutase, total antioxidant capacity, catalase and glutathione peroxidase. The level of oxidative stress was further aggravated after combined exposure. The reactive oxygen species level of AML12 exposed to DEHP and MPs in vitro was significantly higher than NC group, and the combined exposure was significantly higher than the single exposure. The in vivo and in vitro also confirmed that DEHP and MPs could significantly increase the mRNA and protein levels of apoptosis markers and necroptosis markers and there was an additive effect. After N-acetylcysteine treatment in vitro, the above-mentioned oxidative stress level and cell damage decreased significantly. This study provided a reference for advocating the reduction of the mixed use of plastic products, and provided a basis for preventing the harm of plastic products residues.

Mitoquinone protects against acetaminophen-induced liver injury in an FSP1-dependent and GPX4-independent manner

Mitochondrial oxidative stress has been a crucial mediator in acetaminophen (APAP)-induced hepatotoxicity. MitoQ, an analog of coenzyme Q10, is targeted towards mitochondria and acts as a potent antioxidant. This study aimed to explore the effect of MitoQ on APAP-induced liver injury and its possible mechanisms. To investigate this, CD-1 mice and AML-12 cells were treated with APAP. Hepatic MDA and 4-HNE, two markers of lipid peroxidation (LPO), were elevated as early as 2 h after APAP. Oxidized lipids were rapidly upregulated in APAP-exposed AML-12 cells. Hepatocyte death and mitochondrial ultrastructure alterations were observed in APAP-induced acute liver injury. The in vitro experiments showed that mitochondrial membrane potentials and OXPHOS subunits were downregulated in APAP-exposed hepatocytes. MtROS and oxidized lipids were elevated in APAP-exposed hepatocytes. We discovered that APAP-induced hepatocyte death and liver injury were ameliorated by attenuation of protein nitration and LPO in MitoQ-pretreated mice. Mechanistically, knockdown of GPX4, a key enzyme for LPO defense systems, exacerbated APAP-induced oxidized lipids, but did not influence the protective effect of MitoQ on APAP-induced LPO and hepatocyte death. Whereas knockdown of FSP1, another key enzyme for LPO defense systems, had little effect on APAP-induced lipid oxidation but partially weakened the protection of MitoQ on APAP-induced LPO and hepatocyte death. These results suggest that MitoQ may alleviate APAP-evoked hepatotoxicity by eliminating protein nitration and suppressing hepatic LPO. MitoQ prevents APAP-induced liver injury partially dependent of FSP1 and independent of GPX4.

Regulation of BCRP expression and sulfasalazine pharmacokinetics by the nuclear receptor REV-ERBα

BCRP (breast cancer resistance protein) is a crucial efflux transporter involved in the regulation of the pharmacokinetics and pharmacodynamics of a wide range of drugs. Herein, we aimed to investigate a potential role for the nuclear receptor REV-ERBα in the regulation of BCRP expression and sulfasalazine (a BCRP probe substrate) pharmacokinetics. Regulation of BCRP expression by REV-ERBα was assessed using Rev-erbα -/- mice and AML12 and CT26 cells. Pharmacokinetic analysis was performed with Rev-erbα -/- and wild-type mice after sulfasalazine administration. We found that the expression levels of BCRP mRNA and protein were downregulated in the liver and small intestine of Rev-erbα-dificient mice. In line with this, Rev-erbα ablation increased the systemic exposures of oral sulfasalazine. Positive regulation of BCRP expression and function by REV-ERBα was furtherly confirmed in AML12 and CT26 cells. Moreover, indirect regulation of Bcrp expression by REV-ERBα was potentially mediated by a negative transcription factor DEC2, which is a downstream target of REV-ERBα. In conclusion, REV-ERBα positively regulates BCRP expression in mice, thereby affecting sulfasalazine pharmacokinetics.

MiR-194 Up-Regulates Cholesterol 7 Alpha-Hydroxylase Expression via β-Catenin Signaling and Aggravates Cholestatic Liver Diseases

miR-194 is abundantly expressed in hepatocytes, and its depletion increases hepatic resistance to acetaminophen-induced acute injuries. In this study, the biological role of miR-194 in cholestatic liver injury was investigated by using miR-194/miR-192 cluster liver-specific knockout (LKO) mice, in which no liver injuries or metabolic disorders were predisposed. Bile duct ligation (BDL) and 1-naphthyl isothiocyanate (ANIT) were applied to LKO and matched control wild-type (WT) mice to induce hepatic cholestasis. Periportal liver damage, mortality rate, and liver injury biomarkers in LKO mice were significantly less than in WT mice after BDL and ANIT injection. Intrahepatic bile acid level was significantly lower in the LKO liver within 48 hours of BDL- and ANIT-induced cholestasis compared with WT. Western blot analysis showed that β-catenin (CTNNB1) signaling and genes involved in cellular proliferation were activated in BDL- and ANIT-treated mice. The expression levels of cholesterol 7 alpha-hydroxylase (CYP7A1), pivotal in bile synthesis, and its upstream regulator hepatocyte nuclear factor 4α were reduced in primary LKO hepatocytes and liver tissues compared with WT. The knockdown of miR-194 using miRNA inhibitors reduced CYP7A1 expression in WT hepatocytes. In contrast, the knockdown of CTNNB1 and overexpression of miR-194, but not miR-192, in LKO hepatocytes and AML12 cells increased CYP7A1 expression. In conclusion, the results suggest that the loss of miR-194 ameliorates cholestatic liver injury and may suppress CYP7A1 expression via activation of CTNNB1 signaling.

Canagliflozin ameliorates the development of NAFLD by preventing NLRP3-mediated pyroptosis through FGF21-ERK1/2 pathway.

Recent studies have suggested that sodium-glucose co-transporter2 inhibitors go beyond their glycemic advantages to ameliorate the development of NAFLD. However, little research has been done on the underlying mechanisms. Here, we took deep insight into the effect of canagliflozin (CANA), one of the sodium-glucose co-transporter2 inhibitor, on the progression of NAFLD, and explored the molecular mechanisms. Our findings showed that CANA-treated ob/ob and diabetic mice developed improved glucose and insulin tolerance, although their body weights were comparable or even increased compared with the controls. The CANA treatment ameliorated hepatic steatosis and lipid accumulation of free fatty acid-treated AML12 cells, accompanied by decreased lipogenic gene expression and increased fatty acid β oxidation-related gene expression. Furthermore, inflammation and fibrosis genes decreased in the livers of CANA-treated ob/ob and diabetic mice mice. FGF21 and its downstream ERK1/2/AMPK signaling decreased, whereas NLRP3-mediated pyroptosis increased in the livers of the ob/ob and diabetic mice mice, which was reversed by the CANA treatment. In addition, blocking FGF21 or ERK1/2 activity antagonized the effects of CANA on NLRP3-mediated pyroptosis in lipopolysaccharide plus nigericin-treated J774A.1 cells. We conclude that CANA treatment alleviated insulin resistance and the progression of NAFLD in ob/ob and diabetic mice mice independent of the body weight change. CANA protected against the progression of NAFLD by inhibiting NLRP3-mediated pyroptosis and enhancing FGF21-ERK1/2 pathway activity in the liver. These findings suggest the therapeutic potential of sodium-glucose co-transporter2 inhibitors in the treatment of NAFLD.

Glucokinase Inactivation Ameliorates Lipid Accumulation and Exerts Favorable Effects on Lipid Metabolism in Hepatocytes

Glucokinase-maturity onset diabetes of the young (GCK-MODY) is a kind of rare diabetes with low incidence of vascular complications caused by GCK gene inactivation. This study aimed to investigate the effects of GCK inactivation on hepatic lipid metabolism and inflammation, providing evidence for the cardioprotective mechanism in GCK-MODY. We enrolled GCK-MODY, type 1 and 2 diabetes patients to analyze their lipid profiles, and found that GCK-MODY individuals exhibited cardioprotective lipid profile with lower triacylglycerol and elevated HDL-c. To further explore the effects of GCK inactivation on hepatic lipid metabolism, GCK knockdown HepG2 and AML-12 cell models were established, and in vitro studies showed that GCK knockdown alleviated lipid accumulation and decreased the expression of inflammation-related genes under fatty acid treatment. Lipidomic analysis indicated that the partial inhibition of GCK altered the levels of several lipid species with decreased saturated fatty acids and glycerolipids including triacylglycerol and diacylglycerol, and increased phosphatidylcholine in HepG2 cells. The hepatic lipid metabolism altered by GCK inactivation was regulated by the enzymes involved in de novo lipogenesis, lipolysis, fatty acid β-oxidation and the Kennedy pathway. Finally, we concluded that partial inactivation of GCK exhibited beneficial effects in hepatic lipid metabolism and inflammation, which potentially underlies the protective lipid profile and low cardiovascular risks in GCK-MODY patients.

DMF-Activated Nrf2 Ameliorates Palmitic Acid Toxicity While Potentiates Ferroptosis Mediated Cell Death: Protective Role of the NO-Donor S-Nitroso-N-Acetylcysteine

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease that can develop into an aggressive form called nonalcoholic steatohepatitis (NASH), which ultimately progresses to cirrhosis, hepatocellular carcinoma (HCC), and end-stage liver failure. Currently, the deterioration of NAFLD is attributed to specific lipid toxicity which could be due to lipotoxicity and/or ferroptosis. In the current study, we evaluated the involvement of the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf-2), which is a main activator of phase II metabolism in the two types of lipid-induced toxicity in hepatocytes, lipotoxicity by saturated fatty acids, and in ferroptosis, and the effect of NO donor treatment. AML12 cells were exposed to 600 μM palmitic acid to induce lipotoxicity or treated with 20 μM erastin or 5 μM RSL3 for ferroptosis. In SFA-lipotoxicity, pretreatment with the Nrf2 activator dimethyl fumarate (DMF) managed to ameliorate the cells and the oxidative stress level while aggravating ferroptosis due to emptying the thiol pool. On the other hand, the nitric oxide (NO)-donor, S-nitroso-N-acetylcysteine (NAC-SNO) proved to be effective in the prevention of hepatocytes ferroptosis.

Anti-miR-96 and Hh pathway inhibitor MDB5 synergistically ameliorate alcohol-associated liver injury in mice

Alcohol-associated liver disease (ALD) and its complications are significant health problems worldwide. Several pathways in ALD are influenced by alcohol that drives inflammation, fatty acid metabolism, and fibrosis. Although miR-96 has become a key regulator in several liver diseases, its function in ALD remains unclear. In contrast, sonic hedgehog (SHH) signaling has a well-defined role in liver disease through influencing the activation of hepatic stellate cells (HSCs) and the inducement of liver fibrosis. In this study, we investigated the expression patterns of miR-96 and Hh molecules in mouse and human liver samples. We showed that miR-96 and Shh were upregulated in ethanol-fed mice. Furthermore, alcoholic hepatitis (AH) patient specimens also showed upregulated FOXO3a, TGF-β1, SHH, and GLI2 proteins. We then examined the effects of Hh inhibitor MDB5 and anti-miR-96 on inflammatory and extracellular matrix (ECM)-related genes. We identified FOXO3 and SMAD7 as direct target genes of miR-96. Inhibition of miR-96 decreased the expression of these genes in vitro in AML12 cells, HSC-T6 cells, and in vivo in ALD mice. Furthermore, MDB5 decreased HSCs activation and the expression of ECM-related genes, such as Gli1, Tgf-β1, and collagen. Lipid nanoparticles (LNPs) loaded with the combination of MDB5, and anti-miR-96 ameliorated ALD in mice. Our study demonstrated that this combination therapy could serve as a new therapeutic target for ALD.

LncRNA‑miRNA‑mRNA network in female offspring born from obese dams.

Maternal obesity is associated with disturbance of lipid metabolism and obesity in offspring; however, the pathogenesis is still unclear. The present study elucidated the role of potential lipid metabolism-associated long non-coding RNA (lncRNA) and identified the pathways involved in mice born to obese dams. In the present study, maternal obesity was induced by feeding a high-fat diet for 10 weeks in female C57/BL6 mice, whereas control mice were fed a standard diet. All female mice mated with healthy male mice and were allowed to deliver spontaneously. The results demonstrated that female offspring from obese dams presented a tendency to become overweight in the first 8 weeks after birth; however, maternal obesity did not significantly alter the body weight of male offspring. RNA-sequencing analysis was performed on female offspring liver at 3 weeks old. Significantly dysregulated lncRNAs and downstream targets in female offspring liver were identified using bioinformatics analysis. lncRNA, microRNA (miRNA or miR) and mRNA expression levels in liver and AML12 cells were assessed using reverse transcription-quantitative PCR. A total of 8 upregulated and 17 downregulated lncRNAs were demonstrated in offspring from obese dams and lncRNA Lockd was indicated to be a key dysregulated lncRNA. Competing endogenous RNA (ceRNA) models suggested that the lncRNA Lockd/miR-582-5p/Elovl5 pathway was key for lipid metabolism in the liver of offspring from obese dams. Finally, small interfering RNA and miRNA inhibitor transfection was used to evaluate the ceRNA models in AML12 cells. Taken together, the results of the present study indicated that lncRNA Lockd-miR-582-5p-Elovl5 network may be disrupted in lipid metabolism and lead to obesity in the offspring of obese dams. This research will provide new insights into the molecular mechanism of obesity and lipid metabolism disorder.

Self-Propelled Ultrasmall AuNPs-Tannic Acid Hybrid Nanozyme with ROS-Scavenging and Anti-Inflammatory Activity for Drug-Induced Liver Injury Alleviation.

Developing nanomedicines with superior reactive oxygen species (ROS) scavenging capability has emerged as a promising strategy in treating ROS-related diseases, for example, drug-induced liver injury. However, designing nanoscavengers with the self-propelling ability to scavenge ROS actively remains challenging. Here, a self-propelled silica-supported ultrasmall gold nanoparticles-tannic acid hybrid nanozyme (SAuPTB) is designed that can effectively alleviate acetaminophen (APAP)-induced liver injury by scavenging excessive ROS and regulating inflammation. SAuPTB exhibits multienzyme activity and displays significantly enhanced diffusion under hydrogen peroxide (H2 O2 ). This in vitro research shows that SAuPTB can effectively eliminate ROS, increasing the viability of H2 O2 -stimulated cells and reducing the cytotoxicity of APAP/H2 O2 -treated AML12 cells. The in vivo studiesshow that SAuPTB can accumulate at inflammatory sites in mouseliver, resulting in the decrease of alanine aminotransferase, aspartate aminotransferase, and ROS, reduction in pro-inflammatory cytokines and chemokines, hence reduced hepatocyte necrosis, liver injury, and mortality. Furthermore, SAuPTB activates the nuclear erythroid 2-related factor 2 pathway to upregulate antioxidative genes and reduce oxidative stress. Finally, the liver showsdecreased high mobility group box 1 and F4/80+ macrophages, suggesting an anti-inflammatory response. This work provides a novel design strategy of nanozymes for ROS-related disease treatment.

Cross-talk between the RAS-ERK and mTOR signalings-associated autophagy contributes to tripterygium glycosides tablet-induced liver injury

Background and aimsDrug-induced liver injury (DILI) remains a critical issue and a hindrance to clinical application of Tripterygium Glycosides Tablet (TGT) despite its favorable therapeutic efficacy in rheumatoid arthritis. Herein, we aimed to elucidate the molecular mechanisms underlying TGT-induced hepatotoxicity. MethodsChemical profiling of TGT was identified by UPLC-Q/TOF-MS/MS and its putative targets were predicted based on chemical structure similarity calculation. Following “DILI-related gene-TGT putative target” interaction network construction, a list of key network targets was screened according to nodes' topological importance and functional relevance. Both in vivo and in vitro experiments were performed to determine drug hepatotoxicity and the underlying mechanisms. ResultA total of 49 chemical components and 914 putative targets of TGTs were identified. Network calculation and functional modularization screened RAS-ERK and mTOR signalings-associated autophagy to be one of the candidate targets of TGT-induced hepatotoxicity. Experimentally, TGT significantly activated RAS-ERK axis, elevated the number of autophagosomes and the expression of LC3II protein, but reduced the expression of p62 protein and suppressed mTOR phosphorylation in the liver tissues of TGT-induced acute liver injury mice and chronic liver injury mice in vivo and AML12 cells in vitro. Moreover, TGT and mL-098 (an activator of RAS) co-treatment reduced AML12 cell viability via regulating autophagy and TGT-induced liver injury-related indicators more dramatically than TGT treatment alone, whereas Salirasib (an inhibitor of RAS) had an opposite effect. ConclusionRAS-ERK-mTOR cross-talk may play a crucial role in TGT-induced hepatocyte autophagy, offering a promising target for developing novel therapeutics to combat TGT-induced hepatotoxicity.

Comparison of cytotoxic, reactive oxygen species (ROS) and apoptotic effects of propofol, thiopental and dexmedetomidine on liver cells at accumulative doses (AML12).

Propofol, thiopental and dexmedetomidine are hypnotic, sedative, antiepileptic and analgesic agents used in general anesthesia and intensive care. There are many known and yet unknown side effects. Our aim in this study was to examine and compare the cytotoxic, reactive oxygen species (ROS) and apoptotic effects of propofol, thiopental and dexmedetomidine drugs, which are widely used in anesthesia, on liver cells (AML12) in vitro. The half-maximum inhibitory concentration (IC50) doses of the three drugs on AML12 cells were determined using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) method. Then at two different doses of each of the three drugs, apoptotic effects were determined by the Annexin-V method, morphological examinations were determined by acridine orange ethidium bromide method and intracellular reactive oxygen species (ROS) levels were determined by flow cytometry. The IC50 thiopental, propofol and dexmedetomidine doses were found to be 255.008, 254.904 and 34.501 μgr/mL, respectively (p<0.001). The highest cytotoxic effect on liver cells was found in the lowest dose of dexmedetomidine (34.501 μgr/mL) compared to the control group. This was followed by thiopental and propofol, respectively. In this study, propofol, thiopental and dexmedetomidine drugs on AML12 cells were found to have toxic effects by increasing intracellular ROS at two different concentrations higher than clinical doses. It was determined that cytotoxic doses caused an increase in ROS and induced apoptosis in cells. We believe that the toxic effects of these drugs can be prevented by examining the values obtained from this study and the results of future studies.

A small-molecule JNK inhibitor JM-2 attenuates high-fat diet-induced non-alcoholic fatty liver disease in mice.

The prevalence of non-alcoholic fatty liver disease (NAFLD) has been deemed a leading cause of end-stage liver disease. As a member of the mitogen-activated protein kinase family, c-Jun N-terminal kinase (JNK) has been shown to play an important role in the pathogenesis of NAFLD. Here, we identified a novel JNK inhibitor, JM-2, and evaluated its therapeutic effects against NAFLD both in vitro and in vivo. In vitro, JNK was blocked by JM-2 in PA-challenged hepatocytes. C57BL/6 mice were fed a high-fat diet for 6months to develop NAFLD. Mice were treated with JM-2 by intragastric administration. In primary hepatocytes and AML-12 cells, JM-2 treatment significantly suppressed palmitic acid (PA)-induced JNK activation and PA-induced inflammation and cell apoptosis. In addition, JM-2 restricted the production of fibrosis- and lipid metabolism-related genes in PA-challenged hepatocytes. We evaluated the curative effect of JM-2 against NAFLD using a high-fat diet (HFD)-fed mouse model. Based on our findings, JM-2 administration significantly protected the mouse liver from HFD-induced inflammation, lipid accumulation, fibrosis, and apoptosis, accompanied with reduced JNK phosphorylation in the liver tissue. JM-2 affords a significant protective effect against HFD-induced NAFLD by inhibiting JNK activation and is potential to be developed as a candidate drug for NAFLD treatment.

Hepatocyte-derived exosomes deliver H2AFJ to hepatic stellate cells and promote liver fibrosis via the MAPK/STMN1 axis activation.

Hepatic stellate cells (HSCs) activate and acquire proliferative features in response to liver injury. However, mechanisms involved in the activation of fibrotic HSCs remain uncharacterized. This study aims at elaborating the mechanistic basis by which exosomal H2AFJ derived from hepatocytes might affect the activation of HSCs and liver fibrosis. Bioinformatics analysis based on transcriptomic RNA-seq data was used to screen out the downstream regulatory genes and pathways of H2AFJ. Mouse hepatocytes AML-12 cells were stimulated with CCl4 to mimic an in vitro microenvironment of liver fibrosis, from which exosomes were isolated. Next, HSCs were co-cultured with hepatocyte-derived exosomes followed by detection of HSC migration and invasion in the presence of manipulated H2AFJ and STMN1 expression and MAPK pathway inhibitor. It was found that H2AFJ was highly expressed in hepatocyte-derived exosomes after CCl4 stimulation. Hepatocyte-derived exosomal H2AFJ promoted HSC migration and invasion. H2AFJ upregulated c-jun-mediated STMN1 by activating the MAPK signaling pathway. Furthermore, in vivo experiments verified that silencing of H2AFJ attenuated liver fibrosis in mice, while restoration of STMN1 negated its effect. Collectively, hepatocyte-derived exosomal H2AFJ aggravated liver fibrosis by activating the MAPK/STMN1 signaling pathway. This study provides a potential therapeutic target for alleviating liver fibrosis.