Oxidative Stress In Hepatocytes Research Articles

MiRNA-21-5p induces chicken hepatic lipogenesis by targeting NFIB and KLF3 to suppress the PI3K/AKT signaling pathway.

The liver plays a critical role in metabolic activity and is the body's first immune barrier, and maintaining liver health is particularly important for poultry production. MicroRNAs (miRNAs) are involved in a wide range of biological activities due to their capacity as posttranscriptional regulatory elements. A growing body of research indicates that miR-21-5p plays a vital role as a modulator of liver metabolism in various species. However, the effect of miR-21-5p on the chicken liver is unclear. In the current study, we discovered that the fatty liver had high levels of miR-21-5p. Then the qPCR, western blot, flow cytometry, ELISA, dual-luciferase, and immunofluorescence assays were respectively used to determine the impact of miR-21-5p in the chicken liver, and it turned out that miR-21-5p enhanced lipogenesis, oxidative stress, and inflammatory responses, which ultimately induced hepatocyte apoptosis. Mechanically, we verified that miR-21-5p can directly target NFIB and KLF3. Furthermore, our experiments revealed that the suppression of NFIB promoted apoptosis and inflammation, and the KLF3 inhibitor accelerated lipogenesis and enhanced oxidative stress. Furthermore, the co-transfection results suggest that the PI3K/AKT pathway is also involved in the process of miRNA-21-5p-mediate liver metabolism regulation. In summary, our study demonstrated that miRNA-21-5p plays a role in hepatocyte lipogenesis, oxidative stress, inflammation, and apoptosis, via targeting NFIB and KLF3 to suppress the PI3K/AKT signal pathway in chicken.

Lactoferrin as a therapeutic agent for attenuating hepatic stellate cell activation in thioacetamide-induced liver fibrosis

Liver fibrosis is a chronic liver disease caused by prolonged liver injuries. Excessive accumulation of extracellular matrix replaces the damaged hepatocytes, leading to fibrous scar formation and fibrosis induction. Lactoferrin (LF) is a glycoprotein with a conserved, monomeric signal polypeptide chain, exhibiting diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, and antitumoral activities. Previous study has shown LF’s protective role against chemically-induced liver fibrosis in rats. However, the mechanisms of LF in liver fibrosis are still unclear. In this study, we investigated LF’s mechanisms in thioacetamide (TAA)-induced liver fibrosis in rats and TGF-β1-treated HSC-T6 cells. Using ultrasonic imaging, H&E, Masson's, and Sirius Red staining, we demonstrated LF’s ability to improve liver tissue damage and fibrosis induced by TAA. LF reduced the levels of ALT, AST, and hydroxyproline in TAA-treated liver tissues, while increasing catalase levels. Additionally, LF treatment decreased mRNA expression of inflammatory factors such as Il-1β and Icam-1, as well as fibrogenic factors including α-Sma, Collagen I, and Ctgf in TAA-treated liver tissues. Furthermore, LF reduced TAA-induced ROS production and cell death in FL83B cells, and decreased α-SMA, Collagen I, and p-Smad2/3 productions in TGF-β1-treated HSC-T6 cells. Our study highlights LF's ability to ameliorate TAA-induced hepatocyte damage, oxidative stress, and liver fibrosis in rats, potentially through its inhibitory effect on HSC activation. These findings suggest LF's potential as a therapeutic agent for protecting against liver injuries and fibrosis.

Multifaceted Effects of Subchronic Exposure to Chlorfenapyr in Mice: Implications from Serum Metabolomics, Hepatic Oxidative Stress, and Intestinal Homeostasis.

As chlorfenapyr is a commonly used insecticide in agriculture, the health risks of subchronic exposure to chlorfenapyr remained unclear. This study aimed to extensively probe the health risks from subchronic exposure to chlorfenapyr at the NOAEL and 10-fold NOAEL dose in mice. Through pathological and biochemical examinations, the body metabolism, hepatic toxicity, and intestinal homeostasis were systematically assessed. After 12 weeks, a 10-fold NOAEL dose of chlorfenapyr resulted in weight reduction, increased daily food intake, and blood lipid abnormalities. Concurrently, this dosage induced hepatotoxicity and amplified oxidative stress in hepatocytes, a finding further supported in HepG2 cells. Moreover, chlorfenapyr resulted in intestinal inflammation, evidenced by increased inflammatory factors (IL-17a, IL-10, IL-1β, IL-6, IL-22), disrupted immune cells (RORγt, Foxp3), and compromised intestinal barriers (ZO-1 and occludin). By contrast, the NOAEL dose presented less toxicity in most evaluations. Serum metabolomic analyses unveiled widespread disruptions in pathways related to hepatotoxicity and intestinal inflammation, including NF-κB signaling, Th cell differentiation, and bile acid metabolism. Microbiomic analysis showed an increase in Lactobacillus, a decrease in Muribaculaceae, and diminished anti-inflammatory microbes, which further propelled the inflammatory response and leaded to intestinal inflammation. These findings revealed the molecular mechanisms underlying chlorfenapyr-induced hepatotoxicity and intestinal inflammation, highlighting the significant role of the gut microbiota.

Uridine diphosphate glucuronosyltransferase 1A1 prevents the progression of liver injury.

Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in metabolizing and detoxifying endogenous and exogenous substances. However, its contribution to the progression of liver damage remains unclear. To determine the role and mechanism of UGT1A1 in liver damage progression. We investigated the relationship between UGT1A1 expression and liver injury through clinical research. Additionally, the impact and mechanism of UGT1A1 on the progression of liver injury was analyzed through a mouse model study. Patients with UGT1A1 gene mutations showed varying degrees of liver damage, while patients with acute-on-chronic liver failure (ACLF) exhibited relatively reduced levels of UGT1A1 protein in the liver as compared to patients with chronic hepatitis. This suggests that low UGT1A1 levels may be associated with the progression of liver damage. In mouse models of liver injury induced by carbon tetrachloride (CCl4) and concanavalin A (ConA), the hepatic levels of UGT1A1 protein were found to be increased. In mice with lipopolysaccharide or liver steatosis-mediated liver-injury progression, the hepatic protein levels of UGT1A1 were decreased, which is consistent with the observations in patients with ACLF. UGT1A1 knockout exacerbated CCl4- and ConA-induced liver injury, hepatocyte apoptosis and necroptosis in mice, intensified hepatocyte endoplasmic reticulum (ER) stress and oxidative stress, and disrupted lipid metabolism. UGT1A1 is upregulated as a compensatory response during liver injury, and interference with this upregulation process may worsen liver injury. UGT1A1 reduces ER stress, oxidative stress, and lipid metabolism disorder, thereby mitigating hepatocyte apoptosis and necroptosis.

ERRγ-inducible FGF23 promotes alcoholic liver injury through enhancing CYP2E1 mediated hepatic oxidative stress

Fibroblast growth factor 23 (FGF23) is a member of endocrine FGF family, along with FGF15/19 and FGF21. Recent reports showed that under pathological conditions, liver produces FGF23, although the role of hepatic FGF23 remains nebulous. Here, we investigated the role of hepatic FGF23 in alcoholic liver disease (ALD) and delineated the underlying molecular mechanism. FGF23 expression was compared in livers from alcoholic hepatitis patients and healthy controls. The role of FGF23 was examined in hepatocyte-specific knock-out (LKO) mice of cannabinoid receptor type 1 (CB1R), estrogen related receptor γ (ERRγ), or FGF23. Animals were fed with an alcohol-containing liquid diet alone or in combination with ERRγ inverse agonist. FGF23 is mainly expressed in hepatocytes in the human liver, and it is upregulated in ALD patients. In mice, chronic alcohol feeding leads to liver damage and induced FGF23 in liver, but not in other organs. FGF23 is transcriptionally regulated by ERRγ in response to alcohol-mediated activation of the CB1R. Alcohol induced upregulation of hepatic FGF23 and plasma FGF23 levels is lost in ERRγ-LKO mice, and an inverse agonist mediated inhibition of ERRγ transactivation significantly improved alcoholic liver damage. Moreover, hepatic CYP2E1 induction in response to alcohol is FGF23 dependent. In line, FGF23-LKO mice display decreased hepatic CYP2E1 expression and improved ALD through reduced hepatocyte apoptosis and oxidative stress. We recognized CBIR-ERRγ-FGF23 axis in facilitating ALD pathology through hepatic CYP2E1 induction. Thus, we propose FGF23 as a potential therapeutic target to treat ALD.

Albiflorin Alleviates Sepsis-induced Acute Liver Injury through mTOR/p70S6K Pathway.

Sepsis often induces hepatic dysfunction and inflammation, accounting for a significant increase in the incidence and mortality rates. To this end, albiflorin (AF) has garnered enormous interest due to its potent anti-inflammatory activity. However, the substantial effect of AF on sepsis-mediated acute liver injury (ALI), along with its potential mechanism of action, remains to be explored. An LPS-mediated primary hepatocyte injury cell model in vitro and a mouse model of CLP-mediated sepsis in vivo were initially built to explore the effect of AF on sepsis. Furthermore, the hepatocyte proliferation by CCK-8 assay in vitro and animal survival analyses in vivo for the survival time of mice were carried out to determine an appropriate concentration of AF. Then, flow cytometry, Western blot (WB), and TUNEL staining analyses were performed to investigate the effect of AF on the apoptosis of hepatocytes. Moreover, the expressions of various inflammatory factors by ELISA and RT-qPCR analyses and oxidative stress by ROS, MDA, and SOD assays were determined. Finally, the potential mechanism of AF alleviating the sepsis-mediated ALI via the mTOR/p70S6K pathway was explored through WB analysis. AF treatment showed a significant increase in the viability of LPS-inhibited mouse primary hepatocytes cells. Moreover, the animal survival analyses of the CLP model mice group indicated a shorter survival time than the CLP+AF group. AF-treated groups showed significantly decreased hepatocyte apoptosis, inflammatory factors, and oxidative stress. Finally, AF exerted an effect by suppressing the mTOR/p70S6K pathway. In summary, these findings demonstrated that AF could effectively alleviate sepsis-mediated ALI via the mTOR/p70S6K signaling pathway.

Cadmium exacerbates liver injury by remodeling ceramide metabolism: Multiomics and laboratory evidence

Cadmium (Cd) is a toxic heavy metal that primarily targets the liver. Cd exposure disrupts specific lipid metabolic pathways; however, the underlying mechanisms remain unclear. This study aimed to investigate the lipidomic characteristics of rat livers after Cd exposure as well as the potential mechanisms of Cd-induced liver injury. Our analysis of established Cd-exposed rat and cell models showed that Cd exposure resulted in liver lipid deposition and hepatocyte damage. Lipidomic detection, transcriptome sequencing, and experimental analyses revealed that Cd mainly affects the sphingolipid metabolic pathway and that the changes in ceramide metabolism are the most significant. In vitro experiments revealed that the inhibition of ceramide synthetase activity or activation of ceramide decomposing enzymes ameliorated the proapoptotic and pro-oxidative stress effects of Cd, thereby alleviating liver injury. In contrast, the exogenous addition of ceramide aggravated liver injury. In summary, Cd increased ceramide levels by remodeling ceramide synthesis and catabolism, thereby promoting hepatocyte apoptosis and oxidative stress and ultimately aggravating liver injury. Reducing ceramide levels can serve as a potential protective strategy to mitigate the liver toxicity of Cd. This study provides new evidence for understanding Cd-induced liver injury at the lipidomic level and insights into the health risks and toxicological mechanisms associated with Cd.

Investigating the Antioxidant Effect of Deoxyschizandin in Doxorubicin-induced Hepatotoxicity: Role of PTEN Signaling Modulation

Background: The hepatotoxicity of doxorubicin (DOX) is one of its main toxic side effects that cannot be ignored. As an important initiating factor for hepatocyte apoptosis and necrosis, oxidative stress is crucial in DOX-induced hepatotoxicity. Deoxyschizandin (Deo) is a highly effective antioxidant flavonoid that has potential therapeutic potential in liver diseases. Objectives: It is of great clinical significance to demonstrate that Deo can effectively treat DOX-related hepatotoxicity. Materials and methods: The hepatotoxicity animal model was established by DOX, and Deo was used for intervention to observe the therapeutic effect. At the cellular level, phosphatase and tensin homolog deleted on chromosome ten (PTEN) is used as a therapeutic target for Deo to explore its impact on oxidative stress in hepatocytes. Results: Liver function damage and oxidative stress successfully appear in animal bodies. Specific manifestations include biomarkers of liver function damage, abnormal biomarkers of oxidative stress, and excessive production of reactive oxygen species (ROS) in hepatocytes. PTEN signaling plays an important role in Deo’s inhibition of oxidative stress. Conclusion: Our studies indicate that Deo improved DOX-induced liver oxidative stress, whose mechanisms may be related to PTEN signaling.

ACE2 Expressed on Myeloid Cells Alleviates Sepsis-Induced Acute Liver Injury via the Ang-(1-7)-Mas Receptor Axis.

Sepsis-induced acute liver injury (ALI) is common in intensive care units. Angiotensin-converting enzyme 2 (ACE2) plays a vital role in hepatic fibrosis and steatosis; however, its role in sepsis-induced ALI remains unclear. This study found that hepatic ACE2 expression in cecal ligation and puncture (CLP)-treated mice significantly decreased 24h after CLP. ACE2-transgenic (TG) mice exhibited a significant improvement in CLP-induced ALI, accompanied by the inhibition of hepatocyte apoptosis, oxidative stress, and inflammation, while ACE2-knockout mice demonstrated an opposite trend. During sepsis-induced ALI, ACE2-TG could also elevate the Ang-(1-7) and Mas receptor (MasR) levels in liver tissues. Interestingly, the MasR inhibitor A779 abrogated the favorable effects of ACE2 on CLP-induced ALI. In a bone marrow transplantation experiment, the ACE2-TG transplantation group showed significantly improved inflammation and liver dysfunction, less hepatocyte apoptosis, and reduced oxidative stress after CLP compared with the wild-type transplantation group. In contrast, the ACE2-knockout group showed poor inflammatory response and liver dysfunction, significantly more hepatocyte apoptosis, and elevated oxidative stress than the wild-type transplantation group after CLP. ACE2 protects against sepsis-induced ALI by inhibiting hepatocyte apoptosis, oxidative stress, and inflammation via the Ang-(1-7)-Mas receptor axis. Thus, targeting ACE2 may be a promising novel strategy for preventing and treating sepsis-induced ALI.

Human umbilical cord-derived mesenchymal stem cells ameliorate liver fibrosis by improving mitochondrial function via Slc25a47-Sirt3 signaling pathway

Chronic Liver fibrosis may progress to liver cirrhosis and hepatocellular carcinoma (HCC), hence cause a substantial global burden. However, effective therapies for blocking fibrosis are still lacking. Although mesenchymal stem cells (MSCs) have been proven beneficial to liver regeneration after damage, the underlying mechanism of their therapeutic effects are not fully understood. Oxidative stress and mitochondrial functionality alteration directly contributes to the hepatocyte apoptosis and development of liver fibrosis. This study aims to elucidate the mechanism by which hUC-MSC alleviates liver fibrosis and mitochondrial dysfunction. RNA-sequencing was performed to characterize the transcriptomic changes after implantation of hUC-MSCs in mice with liver fibrosis. Next, western blot, RT-PCR, immunohistochemical and immunofluorescence staining were used to evaluate the expression of different genes in vitro and in vivo. Additionally, mitochondrial morphological and dynamic changes, ROS content, and ATP production were examined. Slc25a47, a newly identified liver-specific mitochondrial NAD+ transporter, was notably reduced in CCl4-treated mice and H2O2-stimulated hepatocytes. Conversely, hUC-MSCs increased the Slc25a47 expression and NAD+ level within mitochondria, thereby enhanced Sirt3 protein activity and alleviated mitochondrial dysfunction in the liver. Furthermore, Slc25a47 knockdown could partially abrogate the protective effects of hUC-MSCs on H2O2-induced mitochondrial fission and oxidative stress in hepatocytes. Our study illustrates that Slc25a47 is a key molecular for hUC-MSCs to improve liver fibrosis and regulates mitochondrial function through Sirt3 for the first time, and providing a theoretical basis for the clinical translation of hUC-MSCs transplantation in the treatment of patients with liver fibrosis/cirrhosis.

Orally administered dual-targeted astaxanthin nanoparticles as novel dietary supplements for alleviating hepatocyte oxidative stress.

The enhancement of bioavailability of food bioactive compounds as dietary supplements can be achieved through the development of targeted delivery systems. This study aimed to develop a novel dual-targeted delivery system for hepatocytes and mitochondria using phacoemulsification self-assembly. The delivery systems were engineered by modifying whey protein isolate (WPI) with galactose oligosaccharide (GOS) and triphenylphosphonium (TPP) to improve AXT transport to the liver and promote hepatic well-being. The dual-targeted nanoparticles (AXT@TPP-WPI-GOS) significantly reduced reactive oxygen species in in vitro experiments, thereby slowing down apoptosis. The AXT@TPP-WPI-GOS exhibited a prominent mitochondrial targeting capacity with a Pearson correlation coefficient of 0.76 at 4 h. In vivo pharmacokinetic experiments revealed that AXT@TPP-WPI-GOS could enhance AXT utilization by 28.18 ± 11.69%. Fluorescence imaging in mice demonstrated significantly higher levels of AXT@TPP-WPI-GOS accumulation in the liver compared to that of free AXT. Therefore, these nanoparticles hold promising applications in nutrient fortification, improving the bioavailability of AXT and supporting hepatic well-being.

Kindlin-2 maintains liver homeostasis by regulating GSTP1–OPN-mediated oxidative stress and inflammation in mice

Hepatocyte plays a principal role in preserving integrity of the liver homeostasis. Our recent study demonstrated that Kindlin-2, a focal adhesion protein that activates integrins and regulates cell-extracellular matrix interactions, plays an important role in regulation of liver homeostasis by inhibiting inflammation pathway; however, the molecular mechanism of how Kindlin-2 knockout activates inflammation is unknown. Here we show that Kindlin-2 loss largely downregulates the antioxidant glutathione S-transferase P 1 (GSTP1) in hepatocytes by promoting its ubiquitination and degradation via a mechanism involving protein-protein interaction. This causes overproduction of intracellular reactive oxygen species (ROS) and excessive oxidative stress in hepatocytes. Kindlin-2 loss upregulates osteopontin in hepatocytes partially due to upregulation of ROS and consequently stimulates overproduction of inflammatory cytokines and infiltration in liver. The molecular and histological deteriorations caused by Kindlin-2 deficiency are markedly reversed by systemic administration of an antioxidant N-acetylcysteine in mice. Taken together, Kindlin-2 plays a pivotal role in preserving integrity of liver function.

Integration of proteomics and metabolomics reveals energy and metabolic alterations induced by glucokinase (GCK) partial inactivation in hepatocytes

AimsGlucokinase (GCK) acts as the glucose sensor in maintaining glucose homeostasis. The inactivating mutation of the GCK gene leads to glucokinase-maturity onset diabetes of the young (GCK-MODY). This study aims to gain further insights into the molecular alterations triggered by GCK partial inactivation in hepatocytes, potentially underlying the favorable prognosis of GCK-MODY. Main methodsA GCK knockdown HepG2 cell model was established, and the integration of proteomics and metabolomics was used to gain a comprehensive understanding of the molecular pathway changes caused by GCK inactivation in the liver. Key findingsProteomic analysis identified 257 differential proteins. KEGG pathway enrichment analysis showed that protein expression changes in the GCK knockdown group were significantly enriched in central carbon metabolism, the TCA cycle, amino acid metabolism and the oxidative phosphorylation pathway. Among them, enzymes in the TCA cycle (PC, IDH2, SDH) were significantly downregulated in GCK-knockdown group. Targeted metabolomics revealed that in the GCK knockdown hepatocytes, TCA cycle intermediates were significantly decreased, including pyruvate, oxaloacetate, citrate and succinic acid, and three metabolites increased including glycine, betaine and homocysteine. These metabolic alterations in turn reduced the accumulation of reactive oxygen species in GCK knockdown hepatocytes. Correlation analysis indicated that TCA cycle metabolites were positively correlated with proteins involved in the TCA cycle, carbon metabolism, glycolysis, Ras signaling, fibrosis and inflammation. SignificanceIn conclusion, GCK knockdown reduced TCA cycle flux and oxidative stress in hepatocytes by influencing the levels of key transcription factors and enzymes, providing a comprehensive understanding of the effects of GCK partial inactivation on liver metabolism and molecular mechanisms.

Diabetes-correcting and antioxidant effects of grape pomace extract rich in natural complex of polyphenols

Background. The positive health effects of polyphenols have led to an increased scientific interest in these natural compounds over the past decade. Many studies confirm the effectiveness of polyphenols as additional therapy in diabetes, especially due to the sugar-lowering effect of polyphenols. The aim of the research was to investigate the morphological and functional state of peripheral blood erythrocytes and the indices of oxidative stress in the liver of rats with experimental diabetes and after the administration of grape pomace extract rich in natural complex of polyphenols. Materials and Methods. We obtained grape pomace extract, which contains a variety of polyphenolic compounds. Rats of the following groups were used in the experiments: control animals, animals treated with grape pomace extract rich in natural complex of polyphenols for 14 days, animals with streptozotocin-induced diabetes mellitus, and animals with streptozotocin-induced diabetes mellitus treated with grape pomace extract rich in natural complex of polyphenols for 14 days. The number of erythrocytes and reticulocytes, the concentration of hemoglobin and glycated hemoglobin were determined in the peripheral blood of rats. The activities of catalase, superoxide dismutase and the content of thiobarbituric acid-reactive substance and carbonyl groups of proteins were determined in the liver tissues of rats. Results. The study has shown an increase in the number of erythrocytes and the level of hemoglobin, a decrease in the level of glycated hemoglobin and the number of reticulocytes in the peripheral blood of rats after administration of grape pomace extract rich in natural complex of polyphenols to rats with experimental diabetes. A decrease in the content of thiobarbituric acid-reactive substance and the content of carbonyl groups of proteins of neutral and basic character and an increase in the activity of catalase and superoxide dismutase in liver tissues were found under the same conditions. Conclusions. The results indicate that the extract of the natural complex of polyphenols is capable of correcting the morphological and functional state of erythrocytes, as well as improving the activity of antioxidant enzymes and the content of marker molecules of oxidative stress in hepatocytes of rats under experimental diabetes mellitus.

Cannabidiol protects the liver from α-Amanitin-induced apoptosis and oxidative stress through the regulation of Nrf2

α-Amanitin, the primary lethal toxin of Amanita, specifically targets the liver, causing oxidative stress, hepatocyte apoptosis, and irreversible liver damage. As little as 0.1 mg/kg of α-amanitin can be lethal for humans, and there is currently no effective antidote for α-amanitin poisoning. Cannabidiol is a non-psychoactive natural compound derived from Cannabis sativa that exhibits a wide range of anti-inflammatory, antioxidant, and anti-apoptotic effects. It may play a protective role in preventing liver damage induced by α-amanitin. To investigate the potential protective effects of cannabidiol on α-amanitin-induced hepatocyte apoptosis and oxidative stress, we established α-amanitin exposure models using C57BL/6J mice and L-02 cells in vitro. Our results showed that α-amanitin exposure led to oxidative stress, apoptosis, and DNA damage in both mouse hepatocytes and L-02 cells, resulting in the death of mice. We also found that cannabidiol upregulated the level of Nrf2 and antioxidant enzymes, alleviating apoptosis, and oxidative stress in mouse hepatocytes and L-02 cells and increasing the survival rate of mice. Our findings suggest that cannabidiol has hepatoprotective effects through the regulation of Nrf2 and antioxidant enzymes and may be a potential therapeutic drug for Amanita poisoning.

IRE1α-JNK axis activation contributes to intracellular lipid accumulation in calf hepatocytes

During the perinatal period, dairy cows undergo negative energy balance (NEB), resulting in elevated circulating levels of nonesterified fatty acids (NEFA). Although increased blood NEFA concentrations are a physiological adaptation of early lactation, excessive NEFA in dairy cows is a major cause of fatty liver. Aberrant lipid metabolism leads to hepatic lipid accumulation and subsequently the development of fatty liver. Both IRE1α and JNK have been validated for their association with hepatic lipid accumulation, including their regulatory functions in calf hepatocyte insulin resistance, oxidative stress and apoptosis. Meanwhile, both IRE1α and JNK are involved in lipid metabolism in non-ruminants. Therefore, the aim of this study was to investigate how IRE1α and JNK regulate lipid metabolism in bovine hepatocytes. An experiment was conducted on randomly selected 10 healthy cows (hepatic TG content <1%) and 10 cows with fatty liver (hepatic TG content >5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and β-hydroxybutyrate (BHB) were greater, while serum concentrations of glucose and milk production were lower in cows with fatty liver. The Western blot results revealed that dairy cows with fatty liver had higher phosphorylation levels of c-Jun N-terminal kinase (JNK), c-Jun and inositol-requiring enzyme 1α (IRE1α) in the liver tissue. Three in vitro experiments were conducted using primary calf hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h, which showed that the phosphorylated levels of JNK, c-Jun and IRE1α increased in both linear and quadratic effects. In the second experiment, hepatocytes were treated with high concentrations of NEFA (1.2 mM) for 12 h with or without SP600125, a canonical inhibitor of JNK. Western blot results showed that SP600125 treatment could decrease the expression of lipogenesis-associated proteins (PPARγ and SREBP-1c) and increase the expression of fatty acid oxidation (FAO)-associated proteins (CPT1A and PPARα) in NEFA-treated hepatocytes. The perturbed expression of lipogenesis-associated genes (FASN, ACACA and CD36), and FAO-associated gene ACOX1 were also recovered by JNK inhibition, indicating that JNK reduced excessive NEFA-induced lipogenesis and FAO dysregulation in calf hepatocytes. Third, shRNA targeting IRE1α (sh-IRE1α) was transfected into calf hepatocytes to silence IRE1α, and KIRA6 was used to inhibit the kinase activity of IRE1α. The blockage of IRE1α could at least partially suppressed NEFA-induced JNK activation. Moreover, the blockage of IRE1α downregulated the expression of lipogenesis genes and upregulated the expression of FAO genes in NEFA-treated hepatocytes. In conclusion, these findings indicate that targeting the IRE1α-JNK axis can reduce NEFA-induced lipid accumulation in bovine hepatocytes by modulating lipogenesis and fatty acid oxidation. This may offer a prospective therapeutic target for fatty liver in dairy cows.

EXPRESS: Bifidobacterium bifidum BGN4 fractions ameliorate palmitic acid-induced hepatocyte ferroptosis by inhibiting SREBP1-CYP2E1 pathway.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is strongly associated with disturbances in the intestinal microbiota. Herein, the biological effects and mechanism of Bifidobacterium bifidum BGN4 fractions in regulating hepatocyte ferroptosis during MAFLD progression were investigated. To establish an in vitro model of MAFLD, LO2 cells were subjected to palmitic acid (PA). The mRNA and protein expressions were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. LO2 cell proliferation was examined using MTT and EdU assays, while its apoptosis was evaluated by Flow cytometry. Furthermore, level of reactive oxygen species (ROS) was measured using DCFH-DA staining. Additionally, the levels of Fe2+, malondialdehyde (MDA), and glutathione (GSH), as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were detected using corresponding kits. ChIP and dual-luciferase reporter gene assays were performed to analyze the interaction between SREBP1 and CYP2E1 promoter. Our results revealed that Bifidobacterium bifidum BGN4 fractions effectively ameliorated PA-induced hepatocyte injury, oxidative stress, and ferroptosis. However, these beneficial effects of BGN4 fractions on PA-induced hepatocyte were dramatically reversed by SREBP1 overexpression, suggesting that BGN4 attenuated MAFLD by acting on SREBP1. Moreover, we observed that BGN4 fractions inhibited CYP2E1 transcription by suppressing SREBP1 nuclear translocation. In addition, CYP2E1 overexpression eliminated the inhibitory effect of BGN4 fractions on PA-induced hepatocyte oxidative stress and ferroptosis. These findings collectively indicated that BGN4 fractions reduced CYP2E1 expression by inhibiting SREBP1 nuclear translocation, thereby suppressing hepatocyte oxidative stress and ferroptosis during the development of MAFLD.

The Combined Effect of Copper Nanoparticles and Microplastics on Transcripts Involved in Oxidative Stress Pathway in Rainbow Trout (Oncorhynchus Mykiss) Hepatocytes.

Copper nanoparticles (CuNPs) and microplastics (MPs) are two emerging contaminants of freshwater systems. Despite their co-occurrence in many water bodies, the combined effects of CuNPs and MPs on aquatic organisms are not well-investigated. In this study, primary cultures of rainbow trout hepatocytes were exposed to dissolved Cu, CuNPs, MPs, or a combination of MPs and CuNPs for 48h, and the transcript abundances of oxidative stress-related genes were investigated. Exposure to CuNPs or dissolved Cu resulted in a significant increase in the transcript abundances of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD). Exposure to CuNPs also led to an upregulation in the expression of Na+/K+ ATPase alpha 1 subunit (ATP1A1). Microplastics alone or in combination with CuNPs did not have a significant effect on abundances of the target gene transcripts. Overall, our findings suggested acute exposure to CuNPs or dissolved ions may induce oxidative stress in hepatocytes, and the Cu-induced effect on target gene transcripts was not associated with MPs.

USP14 governs CYP2E1 to promote nonalcoholic fatty liver disease through deubiquitination and stabilization of HSP90AA1

Nonalcoholic fatty liver disease (NAFLD) begins with excessive triglyceride accumulation in the liver, and overly severe hepatic steatosis progresses to nonalcoholic steatohepatitis (NASH), which is characterized by lipid peroxidation, inflammation, and fibrosis. Ubiquitin-specific proteinase 14 (USP14) regulates inflammation, hepatocellular carcinoma and viral infection, but the effect of USP14 on NAFLD is unknown. The aim of this study was to reveal the role of USP14 in the progression of NAFLD and its underlying mechanism. We demonstrated that hepatic USP14 expression was significantly increased in NAFLD in both humans and mice. Hepatic USP14 overexpression exacerbated diet-induced hepatic steatosis, inflammation and fibrosis in mice, in contrast to the results of hepatic USP14 knockdown. Furthermore, palmitic/oleic acid-induced lipid peroxidation and inflammation in hepatocytes were markedly increased by USP14 overexpression but decreased by USP14 knockdown. Notably, in vivo or in vitro data show that USP14 promotes NAFLD progression in a cytochrome p4502E1 (CYP2E1)-dependent manner, which exacerbates hepatocyte oxidative stress, impairs the mitochondrial respiratory chain and inflammation by promoting CYP2E1 protein levels. Mechanistically, we demonstrated by immunoprecipitation and ubiquitination analysis that USP14 inhibits the degradation of heat shock protein 90 alpha family class A member 1 (HSP90AA1) by decreasing its lysine 48-linkage ubiquitination. Meanwhile, upregulation of HAP90AA1 protein promotes CYP2E1 protein accumulation. Collectively, our data indicate that an unknown USP14-HSP90AA1-CYP2E1 axis contributes to NAFLD progression, and we propose that inhibition of USP14 may be an effective strategy for NASH treatment.

Restoration and preservation effects of mung bean antioxidant peptides on H2O2-induced WRL-68 cells via Keap1-Nrf2 pathway.

Mung bean antioxidant peptides (MBAPs) were prepared from mung bean protein hydrolysate, and four peptide sequences including Ser-Asp-Arg-Thr-Gln-Ala-Pro-His (~953 Da), Ser-His-Pro-Gly-Asp-Phe-Thr-Pro-Val (~956 Da), Ser-Asp-Arg-Trp-Phe (~710 Da), and Leu-Asp-Arg-Gln-Leu (~644 Da) were identified. The effects of MBAPs on the oxidation-induced normal human liver cell line WRL-68 were analyzed to determine the mechanism protecting the oxidation-induced injury. The results showed that the cells were subjected to certain oxidative damage by H2O2 induction, as evidenced by decreased cell number and viability, overproduction of intracellular ROS, and decreased mitochondrial membrane potential. Compared with the H2O2-induced group, the MBAP-treated oxidation-induced group exhibited significantly higher cell number and viability, and the intracellular ROS was similar to that of the control group, suggesting that MBAP scavenges excessive intracellular free radicals after acting on the oxidation-induced cells. Combined with Western blotting results, it was concluded that the MBAP-treated oxidation-induced group also significantly promoted the expression of proteins related to the kelch-like ech-related protein 1 (Keap1)/ nuclear factor e2-related factor 2 (Nrf2) signaling pathway, which resulted in an approximately 2-fold increase in antioxidant enzymes, and a decrease in malondialdehyde content of approximately 55% compared to oxidatively-induced cells, leading to the recovery of both cell morphology and viability. These results suggest that MBAPs scavenge intracellular free radicals and improve oxidative stress in hepatocytes through the expression of Keap1/Nrf2 pathway-related protein, thereby reducing oxidative attack on the liver. Therefore, MBAP is applied as a nutritional ingredient in the functional food field, and this study provides a theoretical basis for the high utilization of mung bean proteins.