Effect of mutant β-catenin gene on the proliferation of hepatocytes

AIM:The β-catenin is a key molecule in the Wnt signal pathway, which plays a critical role in normal development and tumorigenesis. However, the mechanisms of the β-catenin on the cell growth control are still not completely defined. The aim of this study was to test the hypothesis that the mutant β-catenin may regulate the hepatocyte proliferation. METHODS: The immortalized murine hepatocyte cell line, AML12, was used for this study. A plasmid that contain mutant β-catenin S33Y was transfected into the AML12 cells and a stable cell line AML12S33Y was established. The cell growth property of this cell line and the parental cell were compared by flow cytometry analysis and direct cell count. The cells were also tested for the ability to form soft agar colonies, and the ability to form tumors in the severe immune deficient mice (SCID). RESULTS:1. The mutant β-catenin containing cell line AML12S33Y has higher proliferating index compared with the parental AML12 cells (P<0.01), suggesting that mutant β-catenin promotes cell growth. 2. The mutant β-catenin cells formed small colonies in soft agar after 4 weeks of culture, but did not generate tumor in SCID mice. CONCLUSION:The mutant β-catenin promotes liver cell growth.     

Effect of GYY4137 on cytosolic lipid decomposition in mouse primary hepatocytes

AIM: To evaluate the effect of GYY4137, a novel hydrogen sulfide (H₂S) donor, on cytosolic lipid decomposition in mouse primary steatosis hepatocytes. METHODS: Oleic acid (OA) was used to induce hepatic steatosis model in vitro. The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion were divided into 4 groups:the cells in control group were incubated with normal medium for 54 h; the cells in model group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 for 6 h; the cells in H₂S group or DL-propar-gylglycine (PAG; an inhibitor of cystathione γ-lysase, inhibiting H₂S synthesis) group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The glycerin release and the protein expression of hormone-sensitive lipase (HSL) in the cells were mea-sured. RESULTS: Compared with model group, the glycerin release and the protein expression of phosphorylated HSL (p-HSL) in H₂S group decreased significantly, while those increased significantly in PAG group. CONCLUSION: In steatosis hepatocytes, exogenous H₂S possibly decreases cytosolic lipid decomposition by decreasing the protein level of p-HSL.     

Effect of exogenous hydrogen sulfide on lipophagy in mouse primary hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) from GYY4137 on lipophagy in mouse primary hepatocytes. METHODS: The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups: the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h; the cells in H₂S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope, phase-contrast microscope or transmission electron microscope. The protein expression of LC3-Ⅰ/Ⅱ in the hepatocytes was determined by Western blot. RESULTS: In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H₂S group increased. CONCLUSION: In steatosis hepatocytes, exogenous H₂S promotes the lipophagy.     

Oxidative stress-activated JNK-MAPK signaling pathway is involved in fluctuant high glucose-induced injury of hepatocytes

AIM: To explore the mechanisms of fluctuant high blood glucose-induced apoptosis of hepatocytes. METHODS: SD rats were randomly divided into normal control group (N), stable high blood glucose group (S), fluctuant high blood glucose group (F) and insulin group (I). Diabetic rats were induced by intraperitoneal injection of streptozotocin (65 mg/kg), and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time points every day. The blood glucose fluctuation patterns of the animals in F group within 12 weeks were similar every day and no significant difference of the HbA1c concentration was observed compared with S group, indicating that the fluctuant hyperglycemia was successfully established in F group. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) and nitric oxide (NO) in the homogenate of the liver tissues were detected by colorimetry. The mRNA and protein levels of JNK, p-JNK, Bax and Bcl-2 were examined by RT-PCR and Western blot. RESULTS: After 12 weeks, the increases in the intakes of food and water, the urine output, and the abnormal liver function were observed in S group, I group and F group. Compared with N group, the MDA level was increased, the content of NO and the activity of SOD and GSH-Px were decreased, and up-regulation of JNK mRNA and p-JNK and Bax proteins, and down-regulation of Bcl-2 were also found in S group, I group and F group. The above effects were more obviously showed in F group. CONCLUSION: Oxidative stress activates JNK-MAPK signaling pathway, which is involved in fluctuant high glucose-induced apoptosis of hepatocytes.     

Lipid-induced p62dok up-regulation enhances hepatic gluconeogenesis

AIM:To investigate the potential role of p62^dok in the regulation of hepatic gluconeogenesis. METHODS:The expression of p62^dok, insulin signaling transduction, and hepatic gluconeogenesis were investigated in the liver tissues of mice treated with high-fat diet(HFD) and in cultured mouse hepatocytes treated with free fatty acid(FFA). The experiments of gene silencing and overexpression were conducted to observe the effects of p62^dok on insulin signal transduction and hepatic gluconeogenesis in cultured mouse hepatocytes. Western blotting was used to detect the protein levels and the phosphorylation statue. RESULTS:The increased p62^dok levels were found in the liver tissues from HFD-treated mice and FFA-treated hepatocytes. Meanwhile, phosphorylation of Akt and forkhead box O1 protein(FoxO1) was were decreased and the expression of glucose-6-phosphatase(G6Pase) and phosphoenolpyruvate carboxykinase(PEPCK) was increased. Silencing of p62^dok in cultured hepatocytes treated with FFA induced the increase in phosphorylation of Akt and FoxO1, and decrease in the protein levels of G6Pase and PEPCK. CONCLUSION:Up-regulation of p62^dok induced by HFD or FFA enhances hepatic gluconeogenesis via inhibiting insulin signal transduction.     

乙酸和β－羟丁酸对体外培养牛肝细胞脂代谢部分关键酶表达的影响

本研究以体外原代培养的奶牛肝细胞为模型,添加不同浓度的乙酸（AcOH）和p羟丁酸（BHBA）,探讨其对奶牛肝细胞脂肪酸代谢关键酶基因表达的影响。添加不同浓度乙酸和β－羟丁酸,培养24h后,提取细胞总RNA。应用实时荧光定量PCR方法,检测脂代谢关键酶长链脂酰辅酶A合成酶1（ACSLl）、柠檬酸合成酶（CS）和乙酰辅酶A羧化酶α（ACCα）mRNA丰度的变化。结果显示,适当浓度的AcOH能够促进肝细胞脂肪酸活化及氧化途径关键酶ACSLl和CS的转录,而高浓度AcOH能够抑制脂肪酸从头合成途径关键酶ACCα的转录;高浓度BHBA能够抑制肝细胞ACSLl、CS和ACCα的转录。说明血液中适当浓度的AcOH能够促进肝脏脂肪酸氧化并抑制脂肪酸从头合成,高浓度BHBA能够抑制肝脏脂氧化和合成,影响乳脂前体物的供应,进而影响乳脂合成。     

Effects of calpain-2 and autophagy-related protein 5 on hepatocyte apoptosis induced by endoplasmic reticulum stress

AIMTo investigate the effects of calpain-2 and autophagy-related protein 5 (Atg5) on apoptosis of BRL-3A rat normal liver cells during endoplasmic reticulum stress (ERS) induced by dithiothreitol (DTT). METH?ODS: BRL-3A cells were treated with DTT at 2.0 mmol/L for 0, 6, 12 and 24 h to induce ERS. Real-time cell analysis (RTCA) was used to measure the effect of DTT on BRL-3A cell proliferation. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mRNA expression of calpain-2 and Atg5 was detected by real-time PCR. The protein levels of calpain-2, Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3) were determined by Western blot. The interaction between calpain-2 and Atg5 was investigated by co-immunoprecipitation (Co-IP). RESULTSThe proliferation of BRL-3A cells treated with DTT was significantly inhibited. The apoptosis of BRL-3A cells was significantly increased after DTT treatment for 6, 12 and 24 h as compared with 0 h group (P<0.05). The cell cycle was arrested in G₁ phase after DTT treatment (P<0.05). After DTT treatment for 6, 12 and 24 h, the mRNA expression of calpain-2 and Atg5 in the BRL-3A cells was significantly increased as compared with 0 h group (P<0.05). The protein levels of calpain-2, Atg12 and Atg7 in the cells treated with DTT for 6, 12 and 24 h were significantly higher than those in 0 h group, and the ratio of LC3-II/LC3-I was also significantly higher than that in 0 h group, while Atg5 expression was significantly lower than that in 0 h group (P<0.05). The results of Co-IP found that the anti-calpain-2 antibody precipitated Atg5 protein from the cell lysates, and the anti-Atg5 antibody also precipitated calpain-2 from the cell lysates, which confirmed the interaction between calpain-2 and Atg5. CONCLUSION Calpain-2 may participate in ERS-induced hepatocyte apoptosis by interacting with Atg5.     

3,4-Dihydroxyacetophenone reduces TG content in L02 cells and hepatic tissue via AMPK pathway

AIM: To investigate the mechanism of 3,4-dihydroxyacetophenone (3,4-DHAP) regulating lipid metabolism in human hepatic cells (L02 cells) and rabbit hepatic tissue. METHODS: L02 cells were divided into normal control group, model group, 3,4-DHAP group and simvastatin group. The cells were collected after treated with drugs for 8 h. Triglyceride (TG) content in the cells was detected by TG kit. RT-qPCR was applied to detect the mRNA expression level of AMP-activated protein kinase (AMPK). The protein levels of AMPK, phosphorylated AMPK (p-AMPK), phosphorylated sterol regulatory element binding protein-1c (p-SREBP-1c) and phosphorylated acetyl-CoA carboxylase (p-ACC) were detected by Western blot. Male New Zealand white rabbits (n=32) were randomized into normal control group, model group, 3,4-DHAP group and simvastatin group. The rabbits were treated with the drugs from week 2 to week 12. At the end of week 12, all rabbits were sacrificed. The liver lipids were measured by oil red O staining, and TG content was analyzed by TG kit. The protein levels of AMPK, p-AMPK, p-SREBP-1c and p-ACC in hepatic tissue were detected by Western blot. RESULTS: In L02 cells, compared with model group, TG content in 3,4-DHAP group was significantly decreased, and the expression of AMPK at mRNA and protein levels and the protein levels of p-AMPK, p-SREBP-1c and p-ACC were significantly increased. In rabbits of 3,4-DHAP group, the TG content was significantly decreased compared with model group, and the protein levels of AMPK, p-SREBP-1c and p-ACC were significantly increased. CONCLUSION: The AMPK signaling pathway may be involved in the mechanism of 3,4-DHAP to reduce TG content in L02 cells and rabbit hepatic tissue.     

Protective effects of somatostatin and octreotide on hepatocytes

AIM: To investigate the protective effect of somatostatin (SST) and octreotide (OCT) on rat hepatocytes. METHODS: The primary hepatocytes were pretreated with different concentrations of SST and OCT. The levels of alanine minotransferase (ALT) and aspartate aminotransferase (AST) in culture supernatant were analyzed by the model of ethanol/carbon tetrachloride (CCl4)-induced hepatocyte injury. Additionally, 75 Sprague-Dawley rats were divided into 5 groups at random, including normal control, model control, SST-treated model groups at high, medium and low doses (200 μg·kg-1·d-1, 100 μg·kg-1·d-1 and 50 μg·kg-1·d-1, respectively). Except for the normal controls, all rats were injected with 40% CCl4 subcutaneously for 8 weeks to establish hepatic fibrosis. Meanwhile, rats of SST-treated model groups were given at different doses of SST twice a day in the same way. Thereafter, the liver function and apoptosis index of hepatocytes were detected by standard enzyme method, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. RESULTS: Compared with those of injury model group, the hepatocytes pretreated with SST (10-8-10-6 mol/L) and OCT (10-7-10-5 mol/L) exhibited significantly decreased levels of ALT and AST in the culture supernatant. Furthermore, most indices of liver function including ALT, AST, alkaline phosphatase (ALP), total bilirubin (TBIL) and albumin (ALB) improved obviously in all SST-treated groups, especially in the group treated with low dose of SST. The apoptosis index of hepatocytes in the fibrotic liver was also reduced greatly by the treatment with low dose of SST. CONCLUSION: SST and OCT may protect hepatocytes against CCl4-induced injury, inhibit hepatocyte apoptosis, and improve the liver function. These findings suggest them a potential efficiency in the prevention of hepatic fibrosis.