Vertical sleeve gastrectomy (VSG) is becoming more and more popular among the world. Despite its dramatic efficacy, however, the mechanism of VSG remains largely undetermined. This study aimed to test interferon (IFN)-γ secretion n of mesenteric lymph nodes in obese mice (ob/ob mice), a model of VSG, and its relationship with farnesoid X receptor (FXR) expression in the liver and small intestine, and to investigate the weight loss mechanism of VSG. The wild type (WT) mice and ob/ob mice were divided into four groups: A (WT+Sham), B (WT+VSG), C (ob/ob+Sham), and D (ob/ob+VSG). Body weight values were monitored. The IFN-γ expression in mesenteric lymph nodes of ob/ob mice pre- and post-operation was detected by flow cytometry (FCM). The FXR expression in the liver and small intestine was detected by Western blotting. The mouse AML-12 liver cells were stimulated with IFN-γ at different concentrations in vitro. The changes of FXR expression were also examined. The results showed that the body weight of ob/ob mice was significantly declined from (40.6±2.7) g to (27.5±3.8) g on the 30th day after VSG (P<0.05). At the same time, VSG induced a higher level secretion of IFN-γ in mesenteric lymph nodes of ob/ob mice than that pre-operation (P<0.05). The FXR expression levels in the liver and small intestine after VSG were respectively 0.97±0.07 and 0.84±0.07 fold of GAPDH, which were significantly higher than pre-operative levels of 0.50±0.06 and 0.48±0.06 respectively (P<0.05). After the stimulation of AML-12 liver cells in vitro by different concentrations of IFN-γ (0, 10, 25, 50, 100, and 200 ng/mL), the relative FXR expression levels were 0.22±0.04, 0.31±0.04, 0.39±0.05, 0.38±0.05, 0.56±0.06, and 0.35±0.05, respectively, suggesting IFN-γ could distinctly promote the FXR expression in a dose-dependent manner in comparison to those cells without IFN-γ stimulation (P<0.05). It was concluded that VSG induces a weight loss in ob/ob mice by increasing IFN-γ secretion of mesenteric lymph nodes, which then increases the FXR expression of the liver and small intestine.
Animals ; Body Weight ; Cell Line ; Gastrectomy ; methods ; Gene Expression ; Hepatocytes ; cytology ; drug effects ; metabolism ; Interferon-gamma ; biosynthesis ; pharmacology ; secretion ; Intestine, Small ; drug effects ; metabolism ; Liver ; drug effects ; metabolism ; Lymph Nodes ; drug effects ; metabolism ; Mesentery ; drug effects ; metabolism ; Mice ; Mice, Obese ; Obesity ; metabolism ; pathology ; surgery ; Receptors, Cytoplasmic and Nuclear ; agonists ; genetics ; metabolism ; Weight Loss

BACKGROUND/AIMS: Chronic liver disease leads to liver fibrosis, and although the liver does have a certain regenerative capacity, this disease is associated with dysfunction of the liver vessels. C-reactive protein (CRP) is produced in the liver and circulated from there for metabolism. CRP was recently shown to inhibit angiogenesis by inducing endothelial cell dysfunction. The objective of this study was to determine the effect of CRP levels on angiogenesis in a rat model of liver dysfunction induced by bile duct ligation (BDL). METHODS: The diameter of the hepatic vein was analyzed in rat liver tissues using hematoxylin and eosin (H&E) staining. The expression levels of angiogenic factors, albumin, and CRP were analyzed by real-time PCR and Western blotting. A tube formation assay was performed to confirm the effect of CRP on angiogenesis in human umbilical vein endothelial cells (HUVECs) treated with lithocholic acid (LCA) and siRNA-CRP. RESULTS: The diameter of the hepatic portal vein increased significantly with the progression of cirrhosis. The expression levels of angiogenic factors were increased in the cirrhotic liver. In contrast, the expression levels of albumin and CRP were significantly lower in the liver tissue obtained from the BDL rat model than in the normal liver. The CRP level was correlated with the expression of albumin in hepatocytes treated with LCA and siRNA-CRP. Tube formation was significantly decreased in HUVECs when they were treated with LCA or a combination of LCA and siRNA-CRP. CONCLUSION: CRP seems to be involved in the abnormal formation of vessels in hepatic disease, and so it could be a useful diagnostic marker for hepatic disease.
Angiogenic Proteins/genetics/metabolism ; Animals ; Bile Ducts/surgery ; C-Reactive Protein/*analysis/genetics/metabolism ; Cells, Cultured ; Disease Models, Animal ; Hepatic Veins/abnormalities ; Hepatocytes/cytology/metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Lithocholic Acid/pharmacology ; Liver/metabolism/pathology ; Liver Cirrhosis/etiology ; Liver Diseases/metabolism/*pathology ; Male ; Microscopy, Fluorescence ; Mitochondria/drug effects/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Serum Albumin/genetics/metabolism

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca2+ concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.
Animals ; Apoptosis/*drug effects ; Cadmium/*toxicity ; Calcium/metabolism ; Cell Communication/drug effects ; Connexin 43/genetics ; Enzyme Activation/drug effects ; Gap Junctions/*drug effects ; Gene Expression Regulation/drug effects ; Hepatocytes/cytology/*drug effects ; Rats ; Signal Transduction/drug effects

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.
Apoptosis ; drug effects ; Cornus ; chemistry ; Endoplasmic Reticulum Stress ; drug effects ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Furaldehyde ; analogs & derivatives ; pharmacology ; Galactosamine ; metabolism ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Liver ; cytology ; drug effects ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; eIF-2 Kinase ; genetics ; metabolism

OBJECTIVETo explore the influence of exogenous putrescine on the function of liver and apoptosis of liver cells in rats.

METHODSNinety healthy clean SD rats were divided into control group (C, n = 10, intraperitoneally injected with 2 mL normal saline), low dosage putrescine group (LP, n = 40), and high dosage putrescine group (HP, n = 40) according to the random number table. Rats in the latter two groups were intraperitoneally injected with approximately 2 mL putrescine (2.5 or 5.0 g/L) with the dosage of 25 or 50 µg/g. Ten rats from group C at post injection hour (PIH) 24 and 10 rats from each of the latter two groups at PIH 24, 48, 72, 96 were sacrificed. Heart blood was obtained for determination of serum contents of ALT and AST. Liver was harvested for gross observation and histomorphological observation with HE staining. Apoptosis was shown with in situ end labeling, and apoptosis index (AI) was calculated. Data among the three groups and those at different time points within one group were processed with one-way analysis of variance or Welch test; LSD or Dunnett's T3 test was used for paired comparison; factorial design analysis of variance of two factors was applied for data between group LP and group HP.

RESULTS(1) No obvious abnormality was observed at gross observation of liver of rats in each group. Liver tissue of rats in group C was normal. Light edema was observed occasionally in liver of rats in groups LP and HP, but necrotic cells were not seen. (2) Content of ALT at PIH 24, 48, 96 and content of AST at PIH 72 and 96 in group LP were respectively (38 ± 10), (45 ± 6), (34 ± 4), (207 ± 18), (196 ± 19) U/L, and content of ALT at PIH 72 and 96 and content of AST at PIH 24, 72, 96 in group HP were respectively (38 ± 6), (48 ± 5), (213 ± 43), (209 ± 40), (230 ± 29) U/L. They were significantly higher than those of rats in group C [(29 ± 5), (163 ± 42) U/L, with P values all below 0.01]. There were statistically significant differences between group LP and group HP in the content of ALT at PIH 48, 72, 96 and content of AST at PIH 96 (with P values all below 0.05). Compared with that at PIH 24 of each group, content of ALT of rats in group LP at PIH 48 and that of rats in group HP at PIH 96, as well as content of AST of rats in group LP at PIH 48, 72, 96 and that of rats in group HP at PIH 48 were significantly increased or decreased (with P values all below 0.05). Factorial analysis showed that the differences due to different concentration of putrescine on content of AST were statistically significant (F = 12.21, P = 0.001), but not on content of ALT (F = 0.01, P = 0.974) between group LP and group HP. (3) AI values of rats in group LP at PIH 24, 48, 72 were respectively (5.69 ± 0.38)%, (13.80 ± 1.66)%, (11.56 ± 1.74)%, and AI values of rats in group HP at PIH 72 and 96 were respectively (10.29 ± 1.43)%, (15.29 ± 1.41)%. They were all obviously higher than AI value of control group at PIH 24 [(3.50 ± 0.30)%, with P values all below 0.01]. There were statistically significant differences between group LP and group HP in AI value at PIH 24, 48, 96 (with P values all below 0.05). Compared with that at PIH 24 of each group, AI value of rats in groups LP and HP at PIH 48, 72, 96 were significantly increased or decreased (with P values all below 0.05). Factorial analysis showed that the differences in the influence of concentration of putrescine and stimulation time on AI value were statistically significant (with F values respectively 22.95 and 130.44, P values all below 0.01).

CONCLUSIONSIntraperitoneal injection of exogenous putrescine in the dosage of 25 or 50 µg/g could lead to certain degree of functional damage of liver and apoptosis of liver cells of rat. The higher the dosage and the longer the stimulation time, the more obvious the damage and apoptosis would be.

Alanine Transaminase ; blood ; Animals ; Apoptosis ; drug effects ; Hepatocytes ; cytology ; drug effects ; Liver ; cytology ; pathology ; Putrescine ; toxicity ; Rats ; Rats, Sprague-Dawley

OBJECTIVEIn the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo.

METHODSHuman cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time.

RESULTSWe observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes.

CONCLUSIONThese studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

Animals ; Cell Line ; Dependovirus ; genetics ; physiology ; Gene Expression ; drug effects ; Genetic Therapy ; Genetic Vectors ; genetics ; physiology ; Hepatocytes ; metabolism ; virology ; Humans ; Liver ; cytology ; metabolism ; virology ; Mice ; Transgenes ; drug effects ; Triterpenes ; pharmacology

OBJECTIVETo observe the effect of Yiguan Decoction (YGD) on differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro.

METHODSRat BMSCs were isolated using whole bone marrow adherent method. The properties of BMSCs were identified by analyzing the expression of surface cytokines by flow cytometry. The third passage cells were differentiated into fat cells to identify their features. BMSCs were incubated with hepatocyte growth factor (HGF) plus fibroblast growth factor 4 (FGF4) or YGD containing serum YGD for 21 days. The mRNA expression of alpha-fetoprotein (alphaAFP), albumin (Alb), and hepatocyte nuclear factor 4alpha (HNF4alpha) were detected by real time PCR. Expression of AFP and cytokeratin 18 (CK18) protein was detected by cell immunofluorescence. Glycogen synthesis was observed using periodic acid-Schiff stain (PAS). CK18, Wnt 3alpha, and alphacatenin protein expressions were detected by Western blot.

RESULTSHigh expression of CD90, CD29, and CD44, and low expression of CD34 and CD11b were observed in BMSCs isolated by whole bone mar- row adherent method, and numerous lipid droplets were observed in BMSCs using oil red O staining. Both YGD containing serum and growth factor stimulated the expression levels of Alb, AFP, HNF4alpha mRNA and CK18 protein. The down-regulated expression of Wnt 3alpha and beta-catenin could be detected at 21 days after induction. The synthesized glycogen granule could be seen. Down-regulated Wnt 3alpha and beta-catenin expression could also be observed.

CONCLUSIONYGD could induce the differentiation of rat BMSCs into hepatocyte-like cells, which was related to down-regulating Wnt/beta-catenin signal pathway.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Hepatocytes ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley ; Wnt Signaling Pathway

Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; Fetal Blood ; cytology ; Hepatocytes ; cytology ; drug effects ; Humans ; Stem Cells ; cytology ; drug effects

Abrus mollis is a widely used traditional Chinese medicine for treating acute and chronic hepatitis, steatosis, and fibrosis. It was found that the total flavonoid C-glycosides from Abrus mollis extract (AME) showed potent antioxidant, anti-inflammatory, and hepatoprotective activities. To further investigate the hepatoprotective effect of AME and its possible mechanisms, lipopolysaccharide (LPS)-induced liver injury models were applied in the current study. The results indicated that AME significantly attenuated LPS-induced lipid accumulation in mouse primary hepatocytes as measured by triglyceride (TG) and total cholesterol (TC) assays and Oil Red O staining. Meanwhile, AME exerted a protective effect on LPS-induced liver injury as shown by decreased liver index, serum aminotransferase levels, and hepatic lipid accumulation. Real-time PCR and immunoblot data suggested that AME reversed the LPS-mediated lipid metabolism gene expression, such as sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase 1 (ACC1). In addition, LPS-induced overexpression of activating transcription factor 4 (ATF4), X-box-binding protein-1 (XBP-1), and C/EBP homologous protein (CHOP) were dramatically reversed by AME. Furthermore, AME also decreased the expression of LPS-enhanced interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2). Here, it is demonstrated for the first time that AME ameliorated LPS-induced hepatic lipid accumulation and that this effect of AME can be attributed to its modulation of hepatic de novo fatty acid synthesis. This study also suggested that the hepatoprotective effect of AME may be related to its down-regulation of unfolded protein response (UPR) activation.
Abrus ; chemistry ; Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Antioxidants ; pharmacology ; therapeutic use ; Chemical and Drug Induced Liver Injury ; drug therapy ; metabolism ; Cholesterol ; metabolism ; Down-Regulation ; Flavonoids ; pharmacology ; therapeutic use ; Glycosides ; pharmacology ; therapeutic use ; Hepatocytes ; drug effects ; metabolism ; Inflammation Mediators ; metabolism ; Lipid Metabolism ; drug effects ; Lipopolysaccharides ; Liver ; cytology ; drug effects ; metabolism ; Male ; Mice, Inbred Strains ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Transaminases ; blood ; Triglycerides ; metabolism ; Unfolded Protein Response ; drug effects

Endoplasmic reticulum stress (ERS) is a new pathway inducing cell apoptosis that has been discovered in recent years. This study focused on the protective effect of Liangxue Huayu recipe (LHR) on tumor necrosis factor-alpha (TNF-alpha) and D-GalN-induced hepatocyte apoptosis. It found that TNF-alpha and D-GalN could obviously inhibit hepatocyte proliferation, induce cell apoptosis, and significantly increase free calcium ions in cytoplasms, as well as protein expressions of ERS apoptosis-related signal molecules phosphorylated PERK, phosphorylated elF2alpha, cleaved Caspase-12, GRP78 and CHOP. After the administration of LHR of different concentrations, compared with the TNF-alpha/GalN injury group, LHR could significantly alleviated L02 hepatocyte proliferation, decreased cell apoptosis, inhibited growth of intracytoplasmic free calcium content, and gradually reduced the protein expressions of phosphorylated PERK, phosphorylated elF2alpha, cleaved Caspase-12, GRP78 and CHOP. These findings indicated that LHR has the inhibitory effect on TNF-alpha and D-GalN-induced hepatocyte apoptosis. Its mechanism may be related to down-regulation of ERS apoptosis-related signal molecules phosphorylated PERK, phosphorylated elF2alpha, cleaved Caspase-12, GRP78 and CHOP that maintain calcium homeostasis in endoplasmic reticulum.
Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; genetics ; metabolism ; Hepatocytes ; cytology ; drug effects ; Humans ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism