Stress-induced Hepatocyte Research Articles

N-glycosylation Modification Reveals Insights into the Oxidative Reactions of Liver in Wuzhishan Pigs

Although porcine liver contributes to their growth and development by nutrition production and energy supply, oxidative stress-induced hepatocyte damage is inevitable during metabolism. N-glycosylation is a common modification in oxidation; nevertheless, the effects of N-glycosylation on pig liver oxidative reactions remain undefined. In this study, liver proteins with N-glycosylation were detected in Wuzhishan (WZS) pigs between 4 and 8 months old and Large White (LW) pigs at 4 months old based on LC-MS/MS. The results showed that the number of differentially expressed proteins (DEPs) was larger between different pig cultivars than that between WZS pigs at various growth periods. The enriched pathways of DEPs were mainly related to oxidative reactions, and 10 proteins were finally selected that primarily consisted of CYPs, GSTs and HSPs with expressions significantly correlating to liver size and weight. The oxidative genes shared N-glycosylation-modified models of N-x-S and N-G. Five out of 10 proteins were upregulated in WZS pigs compared to LW pigs at 4 months old, while five proteins increased in WZS pigs from 4 to 8 months old. In conclusion, this research provides valuable information on the N-glycosylation motifs in liver oxidation genes of WZS pigs.

HSP90-regulated mitophagy can alleviate heat stress damage by inhibiting pyroptosis in the hepatocytes of Wenchang chickens

Heat shock protein 90 (HSP90) has a recognized anti-heat stress injury effect, but its function and corresponding molecular mechanism in heat-stressed hepatocytes are not fully understood, especially in tropical animals. In the present study, we identified several key factors affecting resistance to injury liver tissues from heat-stressed Wenchang chickens (a typical tropical species), such as HSP90, cellular pyroptosis and mitophagy. Heat stress upregulated the NLRP3/Caspase-1/GSDMD-N-mediated cellular pyroptosis pathway and the Pink1/Parkin-mediated mitophagy pathway in chicken hepatocytes, accompanied by the upregulation of HSP90. We also found that HSP90 overexpression significantly reduced heat stress-induced hepatocyte pyroptosis and enhanced mitophagy in primary hepatocytes from Wenchang chickens (PHWCs). HSP90 knockdown significantly increased heat stress-induced hepatocyte pyroptosis and decreased mitophagy in PHWCs. Interestingly, we performed immunoprecipitation and immunofluorescence colocalization and found that HSP90 and Pink1 can interact and directly regulate the level of mitophagy in PHWCs. Our results suggest that HSP90, which regulates Pink1, is an important factor in mitophagy that attenuates heat stress injury by inhibiting cellular pyroptosis.

Quantitative proteomics analysis based on data-independent acquisition reveals the effect of Shenling Baizhu powder (SLP) on protein expression in MAFLD rat liver tissue

BackgroundMetabolic associated fatty liver disease (MAFLD) has become the most common chronic liver disease worldwide, and it is also a high-risk factor for the development of other metabolic diseases. Shenling Baizhu powder (SLP) is a traditional Chinese herbal formula with good clinical efficacy against MAFLD. However, its molecular mechanism for the treatment of MAFLD is still not fully understood. This study used quantitative proteomics analysis to reveal the SLP action mechanism in the treatment of MAFLD by discovering the effect of SLP on protein expression in the liver tissue of MAFLD rats.Materials and methodsQ-Orbitrap LC–MS/MS was used to identify the incoming blood compounds of SLP. The 18 SD male rats were randomly divided into 3 groups (n = 6): control group, HFD group and SLP group. The HFD group and SLP group were established as MAFLD rat models by feeding them a high-fat diet for 4 weeks. Afterwards, the SLP group was treated with SLP (10.89 g/kg/d) for 3 weeks. Biochemical parameters and liver pathological status were measured. Rat liver tissue was analyzed using DIA-based quantitative proteomics and the DEPs were validated by western blotting analysis.ResultsA total of 18 active compounds of SLP were identified and isolated to enter the bloodstream. Comparison of DEPs between control group vs. HFD group and HFD group vs. SLP group revealed that SLP restored the expression of 113 DEPs. SLP catalyzes oxidoreductase activity and binding activity on mitochondria and endoplasmic reticulum to promote lipid oxidative catabolism, maintain oxoacid metabolic homeostasis in vivo and mitigate oxidative stress-induced hepatocyte injury. And 52 signaling pathways including PPAR signaling, arachidonic acid metabolism and glycine, serine and threonine metabolism were enriched by KEGG. PPI topology analysis showed that Cyp4a2, Agxt2, Fabp1, Pck1, Acsm3, Aldh1a1, Got1 and Hmgcs2 were the core DEPs. The western blotting analysis verified that SLP was able to reverse the increase in Fabp1 and Hmgcs2 and the decrease in Pck1 induced by HFD, and the results were consistent proteomic data.ConclusionSLP ameliorates hepatic steatosis to exert therapeutic effects on MAFLD by inhibiting the expression of lipid synthesis genes and inhibiting lipid peroxidation in mitochondria. This study provides a new idea and basis for the study of SLP in the treatment of MAFLD and provides an experimental basis for the clinical application of SLP.

Glycyrrhetinic acid attenuates endoplasmic reticulum stress-induced hepatocyte apoptosis via CHOP/DR5/Caspase 8 pathway in cholestasis

Bile acid (BA)-induced apoptosis is a common pathologic feature of cholestatic liver injury. Glycyrrhetinic acid (GA) is the hepatoprotective constituent of licorice. In the present study, the anti-apoptotic potential of GA was investigated in wild type and macrophage-depleted C57BL/6 mice challenged with alpha-naphthyl isothiocyanate (ANIT), and hepatocytes stimulated with Taurocholic acid (TCA) or Tumor necrosis factor-alpha (TNF-α). Apoptosis was determined by TUNEL positive cells and expression of executioner caspases. Firstly, we found that GA markedly alleviated liver injury, accompanied with reduced positive TUNEL-staining cells, and expression of caspases 3, 8 and 9 in mice modeled with ANIT. Secondly, GA mitigated apoptosis in macrophage-depleted mice with exacerbated liver injury and augmented cell apoptosis. In vitro study, pre-treatment with GA reduced the expression of activated caspases 3 and 8 in hepatocytes stimulated with TCA, but not TNF-α. The ability of GA to ameliorate apoptosis was abolished in the presence of Tauroursodeoxycholic Acid (TUDCA), a chemical chaperon against Endoplasmic reticulum stress (ER stress). Furthermore, GA attenuated the over-expression of Glucose regulated protein 78 (GRP78), and blocked all three branches of Unfolded protein reaction (UPR) in cholestatic livers of mice induced by ANIT. GA also downregulated C/EBP homologous protein (CHOP) expression, accompanied with reduced expression of Death receptor 5 (DR5) and activation of caspase 8 in both ANIT-modeled mice and TCA-stimulated hepatocytes. The results indicate that GA inhibits ER stress-induced hepatocyte apoptosis in cholestasis, which correlates with blocking CHOP/DR5/Caspase 8 pathway.

Oligopeptide of RDPEER from watermelon seeds prevents heat stress-induced liver injury by suppressing oxidative stress and inflammation responses

In this paper, the effect and potential mechanism of supplementary RDPEER (P1, 801 Da), an antioxidant oligopeptide derived from watermelon seeds, on heat stress-induced rat liver injury and HepG2 cell damage were studied. It was observed that supplementation with RDPEER attenuated heat stress-induced hepatocyte injury by increasing HepG2 cell viability, reducing ROS overproduction, improving antioxidant enzyme activity (T-SOD, GSH-Px and CAT) and decreasing MDA content. RDPEER also inhibited HPA axis hyperactivity, serum transaminase activity (AST, ALT) and inflammatory factor levels (INF-γ, IL-6 and TNF-α), activated the endogenous antioxidant pathway (Nrf2/Keap-1), and reduced inflammatory factor transcription (NFκB) and heat shock protein expression (HSP27/72/90). In conclusion, supplementation with RDPEER could enhance antioxidant capacity in vivo and in vitro, reduce the inflammatory and heat shock response to alleviate liver damage caused by heat stress. These findings may provide new insights to alleviate heat stress through nutritional interventions.

Exercise and Metformin Intervention Prevents Lipotoxicity-Induced Hepatocyte Apoptosis by Alleviating Oxidative and ER Stress and Activating the AMPK/Nrf2/HO-1 Signaling Pathway in db/db Mice.

Objective Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2DM) commonly coexist and act synergistically to drive adverse clinical outcomes. This study is aimed at investigating the effects of exercise intervention and oral hypoglycaemic drug of metformin (MET) alone or combined on hepatic lipid accumulation. To investigate if oxidative stress and endoplasmic reticulum stress (ERS) are involved in lipotoxicity-induced hepatocyte apoptosis in diabetic mice and whether exercise and/or MET alleviated oxidative stress or ERS-apoptosis by AMPK-Nrf2-HO-1 signaling pathway. Methods Forty db/db mice with diabetes (random blood glucose ≥ 250 mg/dL) were randomly allocated into four groups: control (CON), exercise training alone (EX), metformin treatment alone (MET), and exercise combined with metformin (EM) groups. Hematoxylin-eosin and oil red O staining were carried out to observe hepatic lipid accumulation. Immunohistochemical and TUNEL methods were used to detect the protein expression of the binding immunoglobulin protein (BiP) and superoxide dismutase-1 (SOD1) and the apoptosis level of hepatocytes. ERS-related gene expression and the AMPK-Nrf2-HO-1 signaling pathway were tested by western blotting. Results Our data showed that db/db mice exhibited increased liver lipid accumulation, which induced oxidative and ER stress of the PERK-eIF2α-ATF4 pathway, and hepatocyte apoptosis. MET combined with exercise training significantly alleviated hepatic lipid accumulation by suppressing BiP expression, the central regulator of ER homeostasis, and its downstream PERK-eIF2α-ATF4 pathway, as well as upregulated the AMPK-Nrf2-HO-1 signaling pathway. Moreover, the combination of exercise and MET displayed protective effects on hepatocyte apoptosis by downregulating Bax expression and TUNEL-positive staining, restoring the balance of cleaved-caspase-3 and caspase-3, and improving the antioxidant defense system to prevent oxidative damage in db/db mice. Conclusion Compared to MET or exercise intervention alone, the combined exercise and metformin exhibited significant effect on ameliorating hepatic steatosis, inhibiting oxidative and ER stress-induced hepatocyte apoptosis via improving the capacity of the antioxidant defense system and suppression of the PERK-eIF2α-ATF4 pathway. Furthermore, upregulation of AMPK-Nrf2-HO-1 signaling pathway might be a key crosstalk between MET and exercise, which may have additive effects on alleviating hepatic lipid accumulation.

Subacute cadmium exposure modulates Th1 polarization to trigger ER stress-induced porcine hepatocyte apoptosis via regulation of miR-369-TNFα axis.

Cadmium (Cd), as a common environmental pollutant, has been reported to cause T lymphocyte dysfunction and cell apoptosis in multiple organs. However, whether subacute Cd exposure can induce apoptosis of hepatocytes and the relationship with Th1/Th2 imbalance were still unclear. In this study, ten 6-week-old piglets were selected and randomly assigned into two groups, the control group and the Cd group. The control group was fed with the standard diet, and for the Cd group, the standard diet was supplemented with 20mg/kg CdCl2; liver tissue was collected on the 40th day of the experiment. Immunofluorescence, qRT-PCR, and western blot were performed to detect the expression of miR-369, Th1/Th2 biomarkers, endoplasmic reticulum (ER) stress-related genes, and apoptotic genes. TUNEL assay was applied to stain apoptotic hepatocytes. In the Cd group, the apoptosis of hepatocytes was significantly increased, and associated with the declined expression of miR-369, Th1 polarization, the elevated expression of ER stress pathway genes and their downstream pro-apoptosis genes, and downregulated expression of anti-apoptotic genes. These results manifest that subacute Cd exposure mediates Th1 polarization to promote ER stress-induced porcine hepatocyte apoptosis via regulating miR-369-TNFα. These results not only provide a basis for the enrichment of Cd toxicology but also support a theoretical foundation for the prevention and therapy of Cd poisoning. Schematic diagram illustrating the proposed mechanism of subacute cadmium exposure modulates Th1 polarization to trigger ER stress-induced porcine hepatocyte apoptosis via regulation of miR-369-TNFα axis.

Senescence in HBV-, HCV- and NAFLD- Mediated Hepatocellular Carcinoma and Senotherapeutics: Current Evidence and Future Perspective.

Simple SummaryRecent scientific discoveries identify cell senescence as pivotal in hepatocellular cancer (HCC) biology. Specifically, hepatitis B virus (HBV), hepatitis C virus (HCV) and non-alcoholic fatty liver disease (NAFLD) are major risk factors for HCC occurrence and it seems that cell senescence serves as a mediator. Furthermore, senescence is also implicated in HCC therapy resistance. Therefore, understanding and harnessing senescence (via senotherapeutics) seems highly important towards the discovery of new preventative and treatment strategies. Herein, we review the role of cell senescence in HBV-, HCV- and NAFLD- mediated HCC, and also explore the possible place of senotherapeutics in the management of HCC. By shining the spotlight on senescence-mediated HCC, we aim to inspire future research towards this rapidly evolving and highly promising field.Cell senescence constitutes a physiological process that serves as protection from malignant transformation of cells. However, recent scientific discoveries also identify cell senescence as pivotal in hepatocellular cancer (HCC) biology. The review herein aimed to accumulate evidence on senescence as a mediator of HCC occurrence in hepatitis B (HBV), C (HCV) virus infections, and non-alcoholic fatty liver disease (NAFLD). In HBV infection, the carcinogenic HBV X protein frequently mutates during chronic infection, and subsequently exhibits different effects on senescence. In HCV infection, senescent non-functional T-cells do not effectively clear pre-malignant hepatocytes. Furthermore, the HCV Core protein inhibits the occurrence of normal stress-induced hepatocyte senescence, allowing damaged cells to maintain their proliferative potential. In NAFLD-mediated HCC, current data point towards the gut microbiome and hepatic stellate cell senescence. Additionally, senescence contributes in the development of resistance in targeted therapies, such as sorafenib. Finally, the promising role of senotherapeutics in HCC was also explored. Overall, although we may still be at a primitive stage in fully unraveling the role of senescence in cancer, it seems that understanding and harnessing senescence may have the potential to revolutionize the way we treat hepatocellular cancer.

Hydrolyzed camel whey protein alleviated heat stress-induced hepatocyte damage by activated Nrf2/HO-1 signaling pathway and inhibited NF-κB/NLRP3 axis.

Liver damage is the most severe complication of heat stress (HS). Hydrolyzed camel whey protein (CWP) possesses bioactive peptides with obviously antioxidant and anti-inflammatory activities. The current study aims to investigate whether CWP that is hydrolyzed by a simulated gastrointestinal digestion process, named S-CWP, protects BRL-3A hepatocytes from HS-induced damage via antioxidant and anti-inflammatory mechanisms. BRL-3A cells were pretreated with S-CWP before being treated at 43°C for 1h, and the levels of the cellular oxidative stress, inflammation, apoptosis, biomarkers for liver function, the activities of several antioxidant enzymes, and the cell viability were analyzed. The expression level of pivotal proteins in correlative signaling pathways was evaluated by western blotting. We confirmed that S-CWP alleviated HS-induced hepatocytes oxidative stress by decreased reactive oxygen species (ROS), nitric oxide (NO), 8-Hydroxy-2'-deoxyguanosine (8-OHdG), lipid peroxidation (LPO), protein carbonylation (PCO), and the activities of NADPH oxidase while enhanced superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) activities, and GSH content. S-CWP suppressed HS-induced inflammatory response by reducing the phosphorylation of NF-κB p65, the expression of NLRP3, and caspase-1 and finally alleviated caspase-3-mediated apoptosis. S-CWP also alleviated HS-induced hepatocyte injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and restoring Heat Shock Protein 70 (HSP70) expression. Furthermore, S-CWP treatment significantly enhanced the expression of NF-E2-related nuclear factor erythroid-2 (Nrf2) and HO-1. The antioxidant and anti-inflammatory effects of S-CWP were weakened by ML385, a specific Nrf2 inhibitor. Additionally, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, significantly reversed S-CWP-induced reduction in the phosphorylation of NF-κB p65. Thus, our results revealed that S-CWP protected against HS-induced hepatocytes damage via activating the Nrf2/HO-1 signaling pathway and inhibiting NF-κB/NLRP3 axis.

Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice Through Endoplasmic Reticulum Stress and Unfolded Protein Response.

The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2−/− mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl4) injection model. We found chronic CCl4 application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2α (eIF2α), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1α/TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2−/− mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl4-treated Lcn2−/− mice. Lcn2−/− mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2−/−, and Chop null (Chop−/−) primary hepatocytes and found Chop−/− hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2α/GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop−/− hepatocytes resumed activation of IRE1α/JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop−/− mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.

Imbalance in mitochondrial dynamics induced by low PGC-1\u03b1 expression contributes to hepatocyte EMT and liver fibrosis

An imbalance in mitochondrial dynamics induced by oxidative stress may lead to hepatocyte epithelial mesenchymal transition (EMT) and liver fibrosis. However, the underlying molecular mechanisms have not been fully elucidated. This study investigated the role of mitochondrial dynamics in hepatocyte EMT and liver fibrosis using an in vitro human (L-02 cells, hepatic cell line) and an in vivo mouse model of liver fibrosis. Findings showed that oxidative stress-induced mitochondrial DNA damage was associated with abnormal mitochondrial fission and hepatocyte EMT. The reactive oxygen species (ROS) scavengers apocynin and mito-tempo effectively attenuated carbon tetrachloride (CCl4)-induced abnormal mitochondrial fission and liver fibrosis. Restoring mitochondrial biogenesis attenuated hepatocyte EMT. Oxidative stress-induced abnormal hepatocyte mitochondrial fission events by a mechanism that involved the down regulation of PGC-1α. PGC-1α knockout mice challenged with CCl4 had increased abnormal mitochondrial fission and more severe liver fibrosis than wild type mice. These results indicate that PGC-1α has a protective role in oxidative stress-induced-hepatocyte EMT and liver fibrosis.

TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway.

Thymic stromal lymphopoietin (TSLP) is a cytokine mainly released by epithelial cells that plays important roles in inflammation, autoimmune disease, and cancer. While TSLP is expressed in the liver at high levels, the role of TSLP in liver ischemia/reperfusion (I/R) injury remains unknown. Experiments were carried out to determine the role of TSLP in liver I/R injury. Wild-type (WT) and TSLP receptor-knockout (TSLPR-/-) mice were subjected to liver partial warm I/R injury. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) level, necrotic areas by liver histology, hepatocyte death, and local hepatic inflammatory responses. Signal pathways were explored in vivo and in vitro to identify possible mechanisms for TSLP in I/R injury. TSLP and TSLPR protein expression increased during liver I/R in vivo and following hepatocyte hypoxia/reoxygenation in vitro. Deletion of TSLPR or neutralization of TSLP with anti-TSLP antibody exacerbated liver injury in terms of serum ALT levels as well as necrotic areas in liver histology. Administration of exogenous recombinant mouse TSLP to WT mice significantly reduced liver damage compared with controls, but failed to prevent I/R injury in TSLPR-/- mice. TSLP induced autophagy in hepatocytes during liver I/R injury. Mechanistically, Akt was activated in WT mice during liver I/R injury. The opposite results were observed in TSLPR-/- mice. In addition, TSLP could directly induce Akt activation in hepatocytes independent of nonparenchymal cells in vitro. Furthermore, the Akt agonist, insulin-like growth factor-1 (IGF-1), prevented I/R injury in TSLPR-/- mice and an Akt inhibitor, LY294002, blocked the protective effects of TSLP in WT mice subjected to I/R. Our data indicate that TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway. Through this pathway, TSLP induces autophagy in hepatocytes. Thus, TSLP is a potent inhibitor of stress-induced hepatocyte necrosis.

Insulin-like growth factor-1 attenuates oxidative stress-induced hepatocyte premature senescence in liver fibrogenesis via regulating nuclear p53\u2013progerin interaction

Stress-induced premature senescence (SIPS), a state of cell growth arrest due to various stimuli, is implicated in the pathogeneses of hepatic fibrogenesis. Progerin, a permanently farnesylated mutant lamin A protein, likely leads to premature senescence to influent liver diseases. The previous reports showed that activation of insulin-like growth factor-1 (IGF-1) signaling could enhance cell longevity and attenuate liver fibrosis. However, the underlying mechanisms about hepatocyte premature senility in liver fibrosis, and how IGF-1 regulates cell premature aging and fibrogenesis, remain poorly understood. In the present study, we found the augment of hepatocyte oxidation and premature aging, along with the decrease of plasm IGF-1 level in patients with liver fibrosis and CCl4-induced liver injury rat models. Nevertheless, IGF-1 gene transfer to CCl4 rats to overexpress intrahepatic IGF-1 relieved hepatocyte oxidative stress and premature senescence, which was likely mediated by the p53/progerin pathway, to improve hepatic steatosis and fibrogenesis. In vitro, H2O2 caused abnormal accumulation of progerin in nuclear and activation of nuclear p53–progerin interaction to trigger primary rat hepatocyte premature senescence through the p21-independent pathway; while these effects were rescued by prolonged exogenous IGF-1 or the IGF-1 adenovirus vector. Furthermore, the IGF-1 adenovirus vector, transfected to H2O2-treated hepatocytes, reversed oxidative stress-induced premature senescence via enhancing cytoplasmic AKT1–p53 interaction and subsequently inhibiting nuclear p53–progerin interaction. Consequently, our data illuminate a novel role of IGF-1 in regulating stress-induced hepatocyte premature senescence in liver fibrosis: prolonged IGF-1 relieves oxidative stress-initiated hepatocyte premature senescence via inhibition of nuclear p53–progerin interaction to ameliorate hepatic steatosis and fibrogenesis.

Autophagy activation by Jiang Zhi Granule protects against metabolic stress-induced hepatocyte injury.

AIMTo elucidate the potential role of autophagy and the protective effects of Jiang Zhi Granule (JZG) in metabolic stress-induced hepatocyte injury.METHODSAn in vitro and in vivo approach was used in this study. HepG2 cells were incubated in culture medium containing palmitate (PA; 0, 0.1, 0.2, 0.3, 0.4 or 0.5 mmol/L) and treated with or without JZG (100 μg/mL) for 24 h or 48 h, and the progression of autophagy was visualized by stable fluorescence-expressing cell lines LC3 and p62. Western blot analyses were performed to examine the expression of LC3-II/LC3-I, p62, mTOR and PI3K, while mitochondrial integrity and oxidative stress were observed by fluorescence staining of JC-1 and reactive oxygen species. C57BL/6 mice were divided into three groups: control group (n = 10), high fat (HF) group (n = 13) and JZG group (n = 13); and, histological staining was carried out to detect inflammation and lipid content in the liver.RESULTSThe cell trauma induced by PA was aggravated in a dose- and time-dependent manner, and hepatic function was improved by JZG. PA had dual effects on autophagy by activating autophagy induction and blocking autophagic flux. The PI3K-AKT-mTOR signaling pathway and the fusion of isolated hepatic autophagosomes and lysosomes were critically involved in this process. JZG activated autophagy progression by either induction of autophagosomes or co-localization of autophagosomes and lysosomes as well as degradation of autolysosomes to protect against PA-induced hepatocyte injury, and protected mitochondrial integrity against oxidative stress in PA-induced mitochondrial dysfunction. In addition, JZG ameliorated lipid droplets and inflammation induced by HF diet in vivo, leading to improved metabolic disorder and associated liver injury in a mouse model of non-alcoholic fatty liver disease (NAFLD).CONCLUSIONMetabolic stress-induced hepatocyte injury exhibited dual effects on autophagy and JZG activated the entire process, resulting in beneficial effects in NAFLD.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression.

AIMTo investigate the protective effect of prostaglandin E1 (PGE1) against endoplasmic reticulum (ER) stress-induced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODSThapsigargin (TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinoma-derived cell line HepG2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RT-PCR. Apoptotic index and cell viability of L02 cells and HepG2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay.RESULTSPretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and HepG2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein (CHOP), glucose-regulated protein (GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and mRNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phospho-eukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A (PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSIONOur study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathway-dependent induction of GRP78 expression.

The constitutive lipid droplet protein PLIN2 regulates autophagy in liver

ABSTRACTExcess triglyceride (TG) accumulation in the liver underlies fatty liver disease, a highly prevalent ailment. TG occurs in the liver sequestered in lipid droplets, the major lipid storage organelle. Lipid droplets are home to the lipid droplet proteins, the most abundant of which are the perilipins (PLINs), encoded by 5 different genes, Plin1 to Plin5. Of the corresponding gene products, PLIN2 is the only constitutive and ubiquitously expressed lipid droplet protein that has been used as a protein marker for lipid droplets. We and others reported that plin2−/− mice have an ∼60% reduction in TG content, and are protected against fatty liver disease. Here we show that PLIN2 overexpression protects lipid droplets against macroautophagy/autophagy, whereas PLIN2 deficiency enhances autophagy and depletes hepatic TG. The enhanced autophagy in plin2−/− mice protects against severe ER stress-induced hepatosteatosis and hepatocyte apoptosis. In contrast, hepatic TG depletion resulting from other genetic and pharmacological manipulations has no effect on autophagy. Importantly, PLIN2 deficiency lowers cellular TG content in wild-type mouse embryonic fibroblasts (MEFs) via enhanced autophagy, but does not affect cellular TG content in atg7−/− MEFs that are devoid of autophagic function. Conversely, adenovirus-shAtg7-mediated hepatic Atg7 knockdown per se does not alter the hepatic TG level, suggesting a more complex regulation in vivo. In sum, PLIN2 guards its own house, the lipid droplet. PLIN2 overexpression protects against autophagy, and its downregulation stimulates TG catabolism via autophagy.

SAT-307 - Tudca Protects against Endoplasmic Reticulum Stress-Induced Inflammasome Activation and Hepatocyte Death in Non-Alcoholic Steatohepatitis

SAT-307 - Tudca Protects against Endoplasmic Reticulum Stress-Induced Inflammasome Activation and Hepatocyte Death in Non-Alcoholic Steatohepatitis

Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure.

ABSTRACTPeroxisome proliferator-activated receptor α (PPARα) is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF). However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress) plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN)- and lipopolysaccharide (LPS)-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA) was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1) PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78), Grp94 and C/EBP-homologous protein (CHOP) in vivo; (2) the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA) treatment reversed liver protection and increased hepatocyte apoptosis; (3) in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF.

삼릉(三稜) 추출물의 간세포 보호 및 미토콘드리아 보호 효과

Objectives : Sparganii Rhizoma is frequently used in traditional herbal medicine for treatment of blood stasis, amenorrhea and functional dyspepsia and has been reported to exhibit anti-oxidant, anti-proliferation and anti-angiogenesis peoperties. In this study, we investigated the cytoprotective effect and underlying mechanism of Sparganii Rhizoma water extract (SRE) against oxidative stress-induced mitochondrial dysfunction and apoptosis in hepatocyte. Methods : To determine the effects of SRE on oxidative stress, we induced synergistic cytotoxicity by co-treatment of arachidonic acid (AA) and iron in the HepG2 cell, a human derived hepatocyte cell line. Results : Treatment of SRE increased relative cell viability and altered the expression levels of apoptosis-related proteins such as Bcl-xL, Bcl-2 and procaspase-3. And SRE also inhibited the mitochondrial dysfunction and excessive reactive oxygen species production induced by AA+iron. In addition, SRE activated of AMP-activated protein kinase (AMPK), a potential target for cytoprotection, by increasing the phosphorylation of AMPKα at Thr-172. Morever, SRE increased phosphorylation of acetyl-CoA carboxylase, a direct downstream target of AMPK. Conclusion : These results indicated that SRE has the ability to protect against oxidative stress-induced hepatocyte damage, which may be mediated with AMPK pathway.

Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model.

Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas’ leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5–12 µM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.