Apoptosis In Liver Research Articles

Gamma-oryzanol prevents ethanol-induced liver injury by ameliorating oxidative stress and modulating apoptosis-related protein expression in mice

Orz provides protection against ethanol-induced liver injury which might be due to its alleviation of oxidative stress and inhibition of apoptosis, possibly inhibiting MAPK signaling pathways mediated mitochondrial signaling pathway activation. • Orz alleviated alcohol-induced oxidative stress and apoptosis in liver. • Orz prevents liver injury by inhibiting MAPK signaling pathway. • Orz prevents liver injury by suppressing mitochondrial apoptotic pathway. Gamma-oryzanol (Orz), a mixture of ferulic acid esters of plant sterols and triterpene alcohols, has been proven to possess multiple activities. The purpose of this study was to explore the hepatoprotective effects and potential mechanisms of Orz against ethanol-induced liver injury in mice. Orz pretreatment significantly reduced the serum levels of ALP, ALT, AST and CYP2E1, and increased the TP and GLB levels in the ethanol-challenged mice. Orz increased the activities of CAT, SOD, GSH-PX and the GSH content in the liver, while decreased hepatic steatosis in ethanol-induced liver injure. Furthermore, Orz significantly decreased the protein expression of ASK1, JNK, P38, active-caspase-9 and active-caspase-3, and the protein ratio of Bax/Bcl-2 in livers of ethanol-challenged mice. These results suggest that Orz provides protection against ethanol-induced liver injury which might be due to its alleviation of oxidative stress and inhibition of apoptosis, possibly inhibiting MAPK signaling pathways mediated mitochondrial signaling pathway activation.

Dipeptidyl peptidase-4 inhibitors and aerobic exercise synergistically protect against liver injury in ovariectomized rats.

Menopause increases the risk of non‐alcoholic fatty liver disease (NAFLD). We investigated the effect of incretin and/ or exercise on the hepatic fat accumulation in ovariectomized rats. Rats were divided into five groups: Group 1: Control rats, Group 2: Ovariectomized rats, Group 3: Ovariectomized rats + Dipeptidyl peptidase‐4 inhibitor (DPPi) (30 mg/kg/day, orally), Group 4: Ovariectomized rats + swimming, and Group 5: Ovariectomized rats + swimming + DPPi. After 6 weeks, Alanine aminotransferase (ALT), glucose, insulin, HOMA IR (Homeostatic Model Assessment for Insulin Resistance), FFA (free fatty acids), Tumor necrosis factor alpha (TNF α), IL6, IL1B levels were measured in blood. The livers were collected for Hematoxylin and eosin (H&E) examination and evaluation of hepatic gene expression of SREBP (sterol regulatory element‐binding protein1c), PPAR α (peroxisome proliferator‐activated receptor alpha), ACC 1 (acetyl‐CoA carboxylase), LC3 (microtubule‐associated protein 1 light chain 3), SIRT (sirtuin), hepatic triglycerides, IL6, IL10, caspase 3 and AMPK (adenosine monophosphate‐activated protein kinase). A significant increase in ALT level and area of liver tissue defects with a significant increase in glucose HOMA IR, serum FFA, IL6, IL1B, TNF α, liver TGs (triglycerides), inflammation, apoptosis, SREBP1c, ACC1 were found in ovariectomized rats as compared to control group with a significant decrease in PPAR α, LC3, AMPK and SIRT1. DPPi treated rats with and without exercise showed a significant improvement in ALT and area of liver tissue defects, inflammation and apoptosis and serum IL6, IL1B, TNF α, FFA, liver LC3, SIRT1, AMPK, TGs, PPAR α, ACC1 and SREBP1c as compared to the ovariectomized group. Findings from the study confirm the derangement of fat metabolism in the ovariectomized rats and showed that incretin‐based therapy and exercise synergistically improved liver fat metabolism, achieved significant beneficial metabolic effects and offer full protection against NAFLD.

Protection from Endotoxin Shock by Selective Targeting of Proinflammatory Signaling to the Nucleus Mediated by Importin Alpha 5.

Endotoxin shock is induced by LPS, one of the most potent virulence factors of the Gram-negative bacteria that cause sepsis. It remains unknown if either proinflammatory stress-responsive transcription factors (SRTFs), ferried to nucleus by importin α5, or lipid-regulating sterol regulatory element binding proteins (SREBPs), transported to the nucleus by importin β1, mediate endotoxin shock. A novel cell-penetrating peptide targeting importin α5 while sparing importin β1 protected 80% of animals from death in response to a high dose of LPS. This peptide suppresses inflammatory mediators, liver glycogen depletion, endothelial injury, neutrophil trafficking, and apoptosis caused by LPS. In d-galactosamine-pretreated mice challenged by 700-times lower dose of LPS, rapid death through massive apoptosis and hemorrhagic necrosis of the liver was also averted by the importin α5–selective peptide. Thus, using a new tool for selective suppression of nuclear transport, we demonstrate that SRTFs, rather than SREBPs, mediate endotoxin shock.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

Prophylactic Fucose can Alleviate Lipopolysaccharide-Induced Cholestatic Liver Injury in Neonatal Rats

Objectives Cholestasis is a common disease of the liver in premature infants and no specific preventive treatment is currently available. Fucose, one of the monosaccharide building blocks of human milk oligosaccharides, may prevent cholestatic hepatic injury by various mechanisms. The aim of this study was to investigate the protective effect of fucose treatment after endotoxin-induced cholestasis in a rat model. Methods Wistar rat pups were divided into four groups as: group I, control group; group II, fucose-supplemented group; group III, lipopolysaccharide (LPS)-administered group, and group IV, LPS-exposed and fucose-supplemented group. Fucose was given 100 mg/kg i.p. every other day between PN5–17. LPS was administered on PN19 to establish endotoxin-induced cholestasis model. On PN21, animals were sacrificed to evaluate liver cell damage and apoptosis. Results Fucose supplementation significantly improved the biochemical parameters that deteriorated in LPS-administered group, significantly increased the expression of bile salt export pump, reduced the number of apoptotic cell death, and greatly prevented LPS-induced cholestatic hepatic injury. Conclusion Given our results, fucose may be useful in reducing hepatic injury and might possess clinical relevance for the preventive treatment of inflammation-induced cholestatic injury in newborns.

Induction of chromosomal and DNA damage and histological alterations by graphene oxide nanoparticles in Swiss mice

The unique physicochemical properties of graphene oxide (GO) nanoparticles increase their uses in a wide range of applications that increase their release into the environment, and thus human exposure. However, the in vivo clastogenicity and genotoxicity of GO nanoparticles have not been well investigated. The current study was, therefore, designed to investigate the possible induction of chromosomal and DNA damage by GO nanoparticles and their impact on the tissue architecture in mice. Oral administration of GO nanoparticles for one or five consecutive days at the three dose levels 10, 20 or 40 mg/kg significantly increased the micronuclei and DNA damage levels in a dose-dependent manner in mice bone marrow cells, as well as caused, histological lesions including apoptosis, necrosis, inflammations and cells degeneration in the mice liver and brain tissue sections compared to the normal control mice. Thus, we concluded that oral administration of GO nanoparticles induced chromosomal and DNA damage in a dose-dependent manner as well as histological injuries in both acute and subacute treatments.

Activation of pCREB/Nrf-2 signaling mediates re-positioning of liraglutide as hepato-protective for methotrexate -induced liver injury (MILI)

Methotrexate (MTX) is commonly used to treat several types of cancer and autoimmune diseases. However, there is increasing concern over its organs toxicities particularly liver toxicity. Liraglutide, a glucagon like peptide-1 agonist, possesses antioxidant and anti-inflammatory features. This study aimed to explore the potential protective effect of liraglutide pre-treatment in ameliorating MTX-induced hepatotoxicity and to further investigate the underlying mechanisms. Rats received 1.2 mg/kg liraglutide intraperitoneal twice daily for 7 days before MTX. Results revealed that liraglutide significantly decreased activities of liver enzymes and oxidative stress in hepatocytes. Furthermore, NF-kB expression and related inflammatory markers (TNF-α, COX-2 and IL-6) were reduced in the pre-treatment group of liraglutide. These data validate the advantageous effects of liraglutide in MTX hepatotoxic animals. In addition, liraglutide increased the expression of the antioxidant transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf-2), along with the transcription of downstream phosphorylated cAMP response element-binding protein (pCREB) which increases the activity of Nrf-2. Additionally, caspase-3 expression/activity and BAX/Bcl-2 ratio were decreased following liraglutide pre-treatment. In conclusion, it was confirmed that liraglutide enhanced the antioxidant activity of liver cells by activating the Nrf-2 and pCREB signaling, thereby, reducing liver cell inflammation and apoptosis induced by MTX.

The ameliorating effect of silymarin against vancomycin-induced apoptosis and inflammation in rat liver

The ameliorating effect of silymarin against vancomycin-induced apoptosis and inflammation in rat liver

Hepatic transcriptome profile of sheep (Ovis aries) in response to overgrazing: novel genes and pathways revealed

BackgroundOvergrazing is a major factor that causes steppe degradation in Inner Mongolian, resulting in extensive ecosystem damage. Scarcity of grass means sheep are smaller and therefore mutton and cashmere production is greatly reduced, which has resulted in massive annual economic losses. Liver is the primary metabolic organ in mammals. It is also the key source of energy supply and detoxification of metabolites in animals, has a close relationship with animal growth. However, investigations on the responses of sheep induced by consequence of overgrazing, particularly those relating to liver-related molecular mechanisms and related metabolic pathways, remain elusive.ResultsThe body weight daily gain of sheep, immune organ indices (liver and spleen), and serum parameters related to immune response, protein synthesis and energy supply (IgG, albumin, glucose and non-esterified fatty acid) were significantly lower in the overgrazing group. Other serum parameters including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, blood urea nitrogen and interleukin-6 were significantly higher in the overgrazing group. For the RNA-Seq results, we identified approximately 50 differentially expressed genes, of which half of were up-regulated and the other half were down-regulated (overgrazing group versus light grazing group). Bioinformatics analysis identified two enriched KEGG pathways including peroxisome proliferator-activated receptor (PPAR) signaling pathway (related to lipolysis) and ECM-receptor interaction (related to liver injury and apoptosis). Additionally, several of the down-regulated genes were related to detoxification and immune response.ConclusionsOverall, based on the high-throughput RNA sequencing profile integrated with the results of serum biochemical analyses, consequences of lower forage availability and quality under overgrazing condition induced altered expression levels of genes participating in energy metabolism (particularly lipid metabolism) and detoxification and immune responses, causing lipolysis and impaired health status, which might be key reasons for the reduced growth performance of sheep. This investigation provides a novel foundation for the development of sheep hepatic gene interactive networks that are a response to the degraded forage availability under overgrazing condition.

Cytochrome b5 Interacts With Cytochrome C and Inhibits Hepatocyte Apoptosis in Brain-dead Rabbit Donors

BackgroundDonation after brain death (BD) liver grafts undergo the process of hypoxia-ischemia, which induces hepatocyte apoptosis, but the underlying mechanisms remain unclear. Cytochrome (Cyt) b5 expression was shown to be low in BD rabbits. This study aimed to investigate if Cyt b5 and Cyt c are involved in liver apoptosis after BD. Methods and resultsLiver tissue samples were obtained from donors after BD and from BD rabbit models. Tissues were analyzed by immunofluorescence, western blotting, and reverse-transcriptase polymerase chain reaction to detect Cyt b5 and Cyt c protein expressions and mRNA. Normal liver cells (LO-2) were cultured under serum deprivation and hypoxia, and analyzed as above. Cyt b5 protein and mRNA levels had decreased, while Cyt c levels had increased in BD liver donors and rabbits. Similar results were obtained in LO-2 cells cultured under hypoxia. After 6 and 12 hours of serum deprivation and hypoxia, apoptosis was increased, the levels of Cyt b5 gradually decreased, and the levels of Cyt c gradually increased over time; meanwhile, the Cyt b5-Cyt c combination was gradually reduced. A negative linear correlation between Cyt b5 and Cyt c was also observed. ConclusionsCyt b5 might be an anti-apoptotic protein that could protect the liver after BD and this protective effect might involve increased binding to Cyt c. This study provides some clues for improving the quality of donor livers.

Bisphenol A induces apoptosis, oxidative stress and inflammatory response in colon and liver of mice in a mitochondria-dependent manner

Bisphenol A (BPA), a widely used industrial compound worldwide, was recently classified as an environmental toxicant. The intestines and liver are responsible for detoxification in humans and animals, and functional damage to these organs adversely affects the health of the body. However, the effect of BPA on intestinal and liver function remains unclear. In this study, we investigated the effects of dietary BPA uptake on oxidative stress, inflammatory response, apoptosis and mitochondrial function in the colons and livers of mice. Dietary BPA uptake significantly reduced the body weights of mice as well as their colon and liver weights. Dietary BPA uptake increased the levels of oxidative stress indicators such as reactive oxygen species, reactive nitrogen species, malondialdehyde and hydrogen peroxide in mouse serum, colon and liver tissues. Antioxidant indicators, such as superoxide dismutase, glutathione peroxidase, catalase and total antioxidant capacity, as well as proinflammatory cytokines (interleukin-1β, interleukin-6, interleukin-8 and tumor necrosis factor-α) were also significantly reduced in the serum, colon, and liver tissues in the BPA group. Moreover, mitochondria-encoded genes and mitochondrial copy numbers were significantly reduced in the colon and liver tissues of the BPA mice. Dietary BPA uptake also increased gene abundance and enzyme activity of caspase-3, -8, -9 and -10. Our study found that dietary BPA induced oxidative stress, inflammatory response, apoptosis and mitochondrial dysfunction in mouse colons and livers.

Palmatine ameliorates nephrotoxicity and hepatotoxicity induced by gentamicin in rats

The aim of this study was to investigate the effects of palmatine on gentamicin toxicity. Rats arranged in four groups: 1- Sham, 2- GM, 3- & 4- GM + palmatine (50 & 100 mg/kg). Gentamicin led to increase in plasma AST, ALT, BUN and creatinine. In addition, fractional excretion of Na and K were increased and urine flow rate and creatinine clearance were decreased in gentamicin group. Liver and renal tissues malondialdehyde were increased, and glutathione was decreased in GM group. TUNEL assay showed induction of apoptosis in liver and kidney in GM group. Palmatine treatment caused reduction in plasma AST, ALT, urine flow rate, creatinine clearance, renal and hapatic malondialdehyde, apoptosis and increase in renal and hapatic glutathione, fractional excretion of Na and K, plasma BUN and creatinine in contrast to GM group. Our data showed palmatine reduced hepatotoxicity and nephrotoxicity by inhibition of oxidative stress and apoptosis.

Maternal undernutrition induces fetal hepatic lipid metabolism disorder and affects the development of fetal liver in a sheep model.

Undernutrition accelerates body fat mobilization to alleviate negative energy balance, which disrupts homeostasis of lipid metabolism in maternal liver. However, little is known about its effect on fetal metabolism and development. Here, a sheep model was used to explore whether maternal undernutrition induces fetal lipid metabolism disorder and further inhibits fetal hepatic development. Twenty pregnant ewes were either fed normally or restricted to 30% level for 15 d, after which fetal hepatic samples were collected to conduct transcriptome, metabolome, histomorphology, and biochemical analysis. Results showed that maternal undernutrition altered the general transcriptome profile and metabolic mode in fetal liver. Fatty acid oxidation and ketogenesis were enhanced in fetal livers of undernourished ewes, which might be promoted by the activated peroxisome proliferator-activated receptor α signaling pathway, whereas cholesterol, steroid, and fatty acid synthesis were repressed. Maternal undernutrition increased triglyceride synthesis, decreased triglyceride degradation, and inhibited phospholipid degradation and synthesis in fetal liver. In addition, our data revealed that maternal undernutrition extremely inhibited DNA replication, cell cycle progression, and antiapoptosis and broke the balance between cell proliferation and apoptosis in fetal liver, indicating that maternal undernutrition affects the growth and development of fetal liver. Generally, these findings provide evidence that maternal undernutrition during pregnancy disturbs fetal lipid metabolism and inhibits fetal hepatic development in sheep, which greatly contribute to the further study of fetal metabolism and development in human beings.-Xue, Y., Guo, C., Hu, F., Zhu, W., Mao, S. Maternal undernutrition induces fetal hepatic lipid metabolism disorder and affects the development of fetal liver in a sheep model.

Stilbene compound trans-3,4,5,4´-tetramethoxystilbene, a potential anticancer drug, regulates constitutive androstane receptor (Car) target genes, but does not possess proliferative activity in mouse liver

The constitutive androstane receptor(CAR) activation is connected with mitogenic effects leading to liver hyperplasia and tumorigenesis in rodents. CAR activators, including phenobarbital, are considered rodent non-genotoxic carcinogens. Recently, trans-3,4,5,4´-tetramethoxystilbene(TMS), a potential anticancer drug (DMU-212), have been shown to alleviate N-nitrosodiethylamine/phenobarbital-induced liver carcinogenesis. We studied whether TMS inhibits mouse Car to protect from the PB-induced tumorigenesis. Unexpectedly, we identified TMS as a murine CAR agonist in reporter gene experiments, in mouse hepatocytes, and in C57BL/6 mice in vivo. TMS up-regulated Car target genes Cyp2b10, Cyp2c29 and Cyp2c55 mRNAs, but down-regulated expression of genes involved in gluconeogenesis and lipogenesis. TMS did not change or down-regulate genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, or Mdm2, which were up-regulated by another Car ligand TCPOBOP. TMS did not increase liver weight and had no significant effect on Ki67 and Pcna labeling indices in mouse liver in vivo. In murine hepatic AML12 cells, we confirmed a Car-independent proapoptotic effect of TMS. We conclude that TMS is a Car ligand with limited effects on hepatocyte proliferation, likely due to promoting apoptosis in mouse hepatic cells, while controlling Car target genes involved in xenobiotic and endobiotic metabolism.

Effect of acute exposure of triazophos on histological structure and apoptosis of the brain and liver of zebrafish (Danio rerio)

Triazophos (TAP) has become a part of widespread pollutant of the aquatic environment due to its residue. Current study was designed to investigate the toxic effect of TAP at different doses (0.06, 0.3 and 1.5 mg/L) to the model organism of zebrafish (Danio rerio) by using multi-endpoint analysis in a 96 h acute exposure test. The direct observation that histological and ultrastructural alteration of zebrafish brain and liver were carried out via paraffin section in hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM), respectively. In addition, a series of methods were applied for exploring the physiological parameters related to cellular apoptosis. Results indicated that vacuolar structure after 96 h treatment with TAP were appeared in the molecular and granular layers of cerebellum. A large number of nuclear retraction, tissues vacuolation and cytoplasmic loss were observed in liver at histological level. From the fine structural level, the mitochondrial vacuolation and membrane damage of brain cells were found and the cristae of mitochondria disintegrated partly in hepatocytes. Onset of such histological structure alterations were one of the most intuitive reflection to TAP exposure, which needs to analyze biochemical alterations for further study. The mitochondrial membrane potential (MMP) showed a downward trend in the brain and liver of zebrafish. Simultaneously, the activity of caspase-3 and caspase-9 increased after 96 h exposure with a concentration-dependent manner, which could be served as a suitable indicator of cellular apoptosis. Furthermore, apoptosis-related genes (Apaf-1, p53, Bax, Bcl-2, caspase-3 and caspase-9) transcription showed different alterations in response to the TAP treatment. These results indicated that TAP exposure led to apoptosis in zebrafish brain and liver and it was speculated that the apoptosis may occur through mitochondrial pathway. The present study demonstrated that the exposure of zebrafish to the insecticide TAP led to observe its effects at both histological structure and apoptosis level in liver and brain.

Hepatic histopathology and apoptosis in diet-induced-obese mice under Escherichia coli pneumonia.

This research was to investigate the difference of hepatic histopathology and apoptosis between the diet-induced obesity (DIO) and normal (lean) mice after Escherichia coli (E. coli) pneumonia. A total of 128 ICR mice were selected to be challenged intranasally with phosphate-buffered saline (PBS) or 4×109CFUs/mL of E. coli, and the liver histopathology and apoptosis were examined pre- and post-infection. Results showed that the liver index, levels of lipid droplets, cytokines, adipocytokines, oxidative stress, apoptotic percentage, and apoptotic related factors in the E. coli-infected mice were generally higher than those in the uninfected mice, whereas the hepatic glycogen and Bcl-2 were the opposite. Interestingly, after E. coli infection, the DIO-E. coli mice exhibited decreased liver index and apoptotic percentages, and reduced levels of TNF-α, IL-6, resistin, MDA, GSH, CAT, Caspase-3, Caspase-9, Bax as well as Bax/Bcl-2 ratio in comparison to the lean-E. coli mice. Our results indicated that E. coli-induced pneumonia caused hepatic histopathological damage, increased hepatic apoptosis, oxidative damages, and higher levels of cytokines and adipocytokines. However, such changes showed less severely in the DIO mice than in the lean mice following E. coli pneumonia.

Triclosan-induced liver injury in zebrafish (Danio rerio) via regulating MAPK/p53 signaling pathway

Long-term exposure of triclosan (TCS), an important antimicrobial agent, can lead to deleterious effects on liver growth and development. However, the related mechanisms on TCS-induced hepatocyte injury remain unclear. Herein, we found that after long-time TCS exposure to adult zebrafish (Danio rerio) from 6 hpf (hours post-fertilization) to 90 dpf (days post-fertilization), the body weight and hepatic weight were significantly increased in concomitant with a large amount of lipid droplet accumulation in liver. Also, TCS exposure resulted in occurrence of oxidative stress by increasing the concentrations of malondialdehyde and reducing the activity of superoxide dismutase both in zebrafish larvae (120 hpf) and adult liver. By H&E staining, we observed a series of abnormal phenomena such as severely hepatocellular atrophy and necrosis, as well as prominently increased hepatic plate gap in TCS-exposure treatment groups. Through AO staining, TCS induced obvious apoptosis in larval heart and liver; through TUNEL assay, a concentration-dependent apoptosis was found to mainly occur in adult liver and its surrounding tissues. The mRNA and protein expression of anti-apoptotic protein Bcl-2 decreased, while that of pro-apoptosis protein Bax significantly increased, identifying that liver injury was closely related to hepatocyte apoptosis. The significant up-regulation of MAPK and p53 at both mRNA and protein levels proved that TCS-induced hepatocyte apoptosis was closely related to activating the MAPK/p53 signaling pathway. These results strongly suggest that long-term TCS-exposure may pose a great injury to zebrafish liver development by means of activating MAPK/p53 apoptotic signaling pathway, also lay theoretical foundation for further assessing TCS-induced ecological healthy risk.

Effects of melanoma differentiation associated gene-7 (MDA-7/IL-24) on apoptosis of liver cancer cells via regulating the expression of B-cell lymphoma-2.

The present study investigated the mechanism of selective killing of liver cancer cells of melanoma differentiation associated gene-7 (MDA-7, also called IL-24α) in order to provide a theoretical basis for gene therapy of liver cancer. A recombinant eukaryotic expression vector (pcDNA3-MDA-7) containing human MDA-7 gene was constructed, which was then delivered to liver cancer cell line HepG2 and normal liver cell line L02. The positive cell clone was screened by G418. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to confirm the occurrence of MDA-7 transcription in the transfected cells. The protein expression of MDA-7 was determined by western blot analysis. The effects of MDA-7 on liver cancer cell proliferation and apoptosis were investigated through MTT assay and flow cytometry by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining. The mitochondrial protein was extracted from the normal liver cell line L02 and liver cancer cell line HepG2 at 3 day post-culture, in which the alterations of anti-apoptotic B-cell lymphoma-2 (Bcl-2), pro-apoptotic Bcl-2 associated X protein (Bax), mitochondria-released cytochrome c and caspase 9 were determined by western blot analysis. pcDNA3-MDA-7 mediated the expression of foreign gene MDA-7 in HepG2 and L02 cells. MDA-7 promoted liver cancer cell apoptosis and inhibited cell proliferation; while no effect was exerted on normal liver cells, as determined by the MTT assay and flow cytometry. Relative to the L02 cells, the protein expression of Bcl-2 was downregulated in the HepG2 cells, while that of Bax, cytochrome c and caspase 9 were upregulated. In the study, the eukaryotic expression vector pcDNA3-MDA-7 was successfully constructed, it can mediate the expression of MDA-7 in human liver cancer cells and normal liver cells and inhibits the proliferation of human liver cancer cells through the restored expression of mitochondrial pro-apoptotic Bcl-2.

Effects of TNFα signaling on iNKT cell development, activation and maturation

Abstract iNKT cells are CD1d-restricted T cells that recognize lipid antigens. Upon activation these cells rapidly produce large amount and a broad spectrum of cytokines. They are important immunoregulatory cells involved in autoimmune disease, liver injury and antitumor immunity. The mechanisms regarding iNKT cell development, activation and maturation have been studied extensively. It is well known that many cytokines and transcription factors are involved in these processes. TNFα, an inflammatory cytokine that can be produced by iNKT cells, co-occurs with iNKT cells in steady state and many disease conditions. How TNFα affects iNKT cell development, activation and maturation is not known. Herein we used a TNFα KO mouse model to address this issue. We found that depletion of TNFα did not affect the frequency of iNKT cells in thymus and spleen, but decreased iNKT cells in liver, and increased iNKT cell apoptosis in liver. The portion of stage-2 (NK1.1loCD44hi) iNKT cells increased, stage-3 (NK1.1hiCD44hi) iNKT cells decreased in the thymus of TNFα KO mice, suggesting that depletion of TNFα impairs iNKT cell development and maturation. The percentage of CD69+ iNKT cells was significantly lower in the thymus, spleen and liver of TNFα KO mice compared to their wild-type littermates, suggesting that depletion of TNFα inhibits iNKT cell activation. Moreover, upon activation, the percentage of splenic IL-4- and IFN-g-producing iNKT cells was significantly lower in TNFα KO mice than in their wild-type littermates. Depletion of TNFα increased PLZF+ iNKT cells in thymus and downregulated the expression of CD122 on iNKT cells. Taken together, TNFα signaling plays important roles in regulation of iNKT cell development, activation and maturation.

The Edible Insect Gryllus bimaculatus Protects against Gut-Derived Inflammatory Responses and Liver Damage in Mice after Acute Alcohol Exposure.

Accumulation of reactive oxygen species (ROS) in response to excess alcohol exposure is a major cause of gut barrier disruption and lipopolysaccharide (LPS)-induced hepatic inflammation, as well as liver steatosis and apoptosis. This study was designed to investigate protective effects of the cricket Gryllus bimaculatus, an edible insect recognized by the Korea Food and Drug Administration, against acute alcoholic liver damage in mice. Administration of G. bimaculatus extracts (GBE) attenuated alcohol-induced steatosis and apoptotic responses in the liver and intestinal permeability to bacterial endotoxin. These protective effects were associated with suppression of ROS-mediated oxidative stress in both the liver and small intestine. Furthermore, in vivo and in vitro studies revealed that GBE inhibits LPS-induced Kupffer cell activation and subsequent inflammatory signaling. Importantly, the protective effects of GBE were more potent than those of silymarin, a known therapeutic agent for alcoholic liver diseases.