肝脏脂肪变,低剂量乙醇 z-bo 2021-11-11 370

　　Introduction: Patients with alcoholic liver disease (ALD) exhibit a wide range of defects, from simple steatosis to steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. In order to study the pathophysiology and treatment of ALD, it is very necessary to establish a suitable animal model. A recent study used adult zebrafish as a model of chronic ethanolic liver disease. The study showed that chronic ethanol treatment (1% ethanol v/v) can cause ethanolic liver disease to develop from simple steatosis to fibrosis after 2-12 weeks of treatment化. However, the concentration of ethanol in the human body is still higher than the lethal concentration (>0.5%). In addition, we have recently discovered that adult zebrafish lose appetite during 1% ethanol treatment, which may affect the pathogenesis of ethanolic liver disease. Another study of adult zebrafish ALD showed that high-dose ethanol treatment (0.5%) for 2-4 weeks is sufficient to induce a range of ALD symptoms, from steatosis to liver damage, including fibrosis. However, these two studies have focused on the pathological analysis of the liver and the changes in the expression of liver marker genes related to inflammation and adipogenesis; there is currently no study on the cellular stress-related genes directly related to liver injury. Therefore, a comprehensive gene expression analysis of adult zebrafish is needed to establish a feasible ALD model. In addition, since blood ethanol concentrations higher than 0.5% are fatal to humans, 1% ethanol treatment to represent chronic ALD may not be ideal. In order to summarize the status of human ALD, we tested low-dose ethanol, which can induce human poisoning. In this study, we explored whether low-dose ethanol (0.2%v/v) can reproduce ethanol-induced liver damage and related metabolic changes in ALD patients, such as increased blood glucose and neutral lipid levels. Importantly, 0.2% ethanol is close to the blood ethanol concentration of a drunk person. To evaluate this metabolic change and liver damage, we measured serum alanine aminotransferase (ALT), glucose and triacylglycerol levels in adult zebrafish after 4 weeks of ethanol exposure, as well as liver histological analysis. In addition, gene expression analysis of genes related to liver inflammation, cell death, fibrosis, lipid and glucose metabolism was performed by quantitative rt-PCR. In addition, in order to identify the two main stress signaling pathways that lead to ethanol-induced liver injury, we evaluated multiple genes related to oxidative stress and endoplasmic reticulum (ER) stress.

　　Materials and methods: Two groups of 20 wild-type male zebrafish (8 months old) were raised in a glass aquarium containing 8 L of water, with or without 0.2% ethanol (V/V) for 4 weeks. Change fresh water with/without ethanol every morning to reduce ethanol concentration, which is affected by fish metabolism and natural evaporation. We feed ordinary food twice a day (10 mg/fish/meal, a test amount that can be completely consumed in 10 minutes).

　　H&E staining: The corpse is fixed in 4% paraformaldehyde at a temperature of 4°C for one night to two days. The fixed samples were embedded in 1.2% agarose/5% sucrose and saturated in 30% sucrose at 4°C for 1 to 2 days. Freeze the fixed block with liquid nitrogen. Prepare 10um slices.

　　Bitter Sour Sirius Red Staining: The liver is fixed in 4% paraformaldehyde at 4°C for one night to two days. The fixed sample was embedded in paraffin and sectioned. For Picric Acid Red staining, the sections are deparaffinized in xylene and hydrated in distilled water. The slides were incubated with 0.1% Sirius Red and Fast Green FCF solution for 1 h, and washed with acidified water (0.5% acetic acid). The sections are cleaned in xylene and fixed with Victamont permanent media.

　　Oil Red O (ORO) staining: ORO staining the cross-section larvae, and drying 10μm thick frozen sections at room temperature for 5min. Drop 150μl of ORO working solution (5% ORO with 60% isopropanol) and stain the slide for 30s, rinse the slide with distilled water, and fix it with 75% glycerol.

　　Blood preparation and biochemical analysis: Minimally invasive blood collection with heparinized needles to obtain zebrafish blood. The blood sampling site is in the dorsal aorta area along the body axis and the back of the anus. Adult zebrafish blood was collected 20 h after rearing, and diluted 1:10 with PBS. The average blood volume of the three fishes (average weight = 0.6 g) was 25 μl. Use Contour Next blood glucose test strips and Bayer Contour Next diabetes EZ meter to measure blood glucose levels. Centrifuge the plasma in a refrigerated centrifuge at 2000×g for 15 min. . Transfer 10 μl of plasma to a 96-well plate, and use a microplate-based alt activity assay kit to determine alt. Use INFINITY? Triglyceride liquid stabilizing reagent to measure triglycerides (TG) in diluted plasma. Three non-ethanol-exposed control fish and ethanol-exposed fish were fasted for one night and then their livers were harvested. Hepatic tissue homogenization buffer (250 mM sucrose, 25 mM KCl, 0.5 mM EDTA, 50 mM Tris HCl, pH 7.4) was homogenized by ultrasound with 1X protease and phosphatase inhibitor. We collected the lysate and used Pierce® BCA protein analysis to determine the protein concentration. Store the lysate at -80°C before analysis. According to the manufacturer's protocol, a triglyceride reagent is used to measure liver TG. "Measurement of intracellular reactive oxygen species: OXISCEAD was used to measure intracellular ROS. Collect the lysate (1 mg/ml), and immediately perform ROS/RNS determination. The fluorescence intensity of peroxide fluorescent dichlorofluorescein (DCF) was detected in 480EX/530μEm BMG LabTeaCalooStAR (Germany), which was formed by the non-fluorescent precursor dichlorodihydrofluorescein (DCFH).

　　Measure GSH and GSSG levels: Enzyme cycling method is used to quantify the content of GSH and GSSG in liver lysate. The protein in the control feed and adult liver extract enriched with PA feed was precipitated with sulfosalicylic acid, and then the supernatant was divided into two tubes. For reduced GSH, the supernatant was incubated with thiol fluorescent probe IV, and the fluorescence intensity was measured at 400Ex/465Em. For total GSH (GSH+GSSG), the supernatant was neutralized with triethanolamine and incubated with the reducing system (containing NADPH and glutathione reductase) at 37°C for 20 min.

　　Result: Low-dose ethanol induces liver damage and mild fibrosis in adult zebrafish: We tested whether low-dose ethanol treatment can induce liver damage in adult zebrafish. Three groups of 14-month-old wild-type zebrafish were isolated in a glass jar with a capacity of 10 liters. The control group was kept in an isolated water tank with system water from the main fish facility. The induction group was raised in water containing 0.2% ethanol (v/v). The ethanol-treated water is replaced every other day for 4 weeks. Histological analysis showed that in the fish treated with 0.2% ethanol, balloon-like hepatocyte accumulation and mild fibrosis appeared. In addition, the measurement of serum alanine aminotransferase (ALT) activity showed that liver damage occurred in the ethanol-treated group compared with the untreated control group. In addition, the gene expression analysis of inflammation, cell death, liver damage, and fibrosis using qPCR showed that compared with untreated siblings, the test genes in the liver of the ethanol treatment group were significantly higher, supporting the serum after chronic ethanol treatment alt rises.

　　Low-dose chronic ethanol induces hyperglycemia and hyperlipidemia in adult zebrafish: long-term drinking causes hyperglycemia and hypertriglyceridemia. We tested whether low-dose chronic ethanol induction of adult zebrafish can reproduce the symptoms observed in humans. A blood sample was collected from the dorsal aorta of the zebrafish trunk with a heparin-coated glass pipette. The results of blood ethanol content measurement showed that the blood ethanol concentration at 1 day and 4 weeks after 0.2% (v/v) ethanol exposure was 0.165% and 0.175%, respectively. We found that the blood glucose level in the 0.2% ethanol treatment group was elevated (1.3 times higher than the untreated group). In addition, we found that after 0.2% ethanol treatment, the level of triacylglycerol was significantly increased (2.2 times) compared to the untreated control group. Therefore, low-dose chronic ethanol treatment of adult zebrafish reproduces the basic metabolic changes in patients with ethanolic liver disease, such as hyperglycemia and hyperlipidemia.

　　The effect of ethanol on liver lipid metabolism and steatosis in zebrafish: In order to study the effect of chronic ethanol treatment on lipid metabolism in adult zebrafish liver, we used qPCR to detect the mRNA expression of lipid metabolism-related genes. We found a significant increase in lipid-producing transcription factor genes, including sterol regulatory element binding transcription factor 1 (srebp1), srebp2 and CCAAT enhanced binding protein α (cebpa). Under the condition of 0.2% ethanol, the genes involved in fatty acid synthesis increased significantly. After ethanol treatment, the expression of acetyl-CoA and malonyl-CoA to produce palmitic acid (acc1) fatty acid synthase (fasn) increased, and triglyceride acyltransferase 2 (dgat2) catalyzed the formation of triglycerides from fatty acids and diglycerides Increased expression. We also studied the expression of genes related to liver fat uptake. In addition, we also found that low-density lipoprotein receptor (ldlr) and lipoprotein lipase (lpl) gene expression increased after ethanol treatment, suggesting an increase in liver lipid uptake induced by ethanol. In addition, ethanol-induced liver steatosis was confirmed by oil red o staining and quantitative determination of intrahepatic triglycerides. We found that total triglycerides increased by 2 times under 0.1% ethanol treatment, and 3 times in liver total triglycerides under 0.2% ethanol treatment. Therefore, low-dose chronic ethanol treatment of adult zebrafish can increase the lipid metabolism and steatosis phenotype of ALD.

　　Ethanol treatment increases gene transcription related to superoxide formation and oxidative stress response: It is well known that chronic ethanol consumption increases reactive oxygen species (ROS) and ROS-mediated oxidative stress in the liver, which may be the main stress leading to liver damage. To solve whether chronic ethanol treatment can induce oxidative stress in zebrafish liver, we analyzed the mRNA expression of genes directly related to superoxide production and oxidative stress. We found that ethanol treatment significantly increased the expression of NADPH oxidases (NOX1, NOX2, and NOX5), which are responsible for the production of superoxide, while reducing NOX4, which is responsible for the production of alkaline hydrogen peroxide in the liver. We speculate that chronic ethanol treatment in zebrafish may increase superoxide levels in the liver. The accumulation of superoxide in the liver can increase the expression of mitochondrial superoxide dismutase (SOD2) mRNA. The increase in SOD2 expression may lead to the accumulation of hydrogen peroxide, which leads to an increase in oxidative stress in the liver. Because oxidative stress increases the transcriptional activation of genes related to oxidative stress, we analyzed two main cellular redox response systems: the glutathione system and the thioredoxin system. After ethanol treatment, the expressions of glutathione reductase (GSR), glutathione S transferase PI 1/2 (GSTP1/2), and glutathione peroxidase 1A/4A (GPX1A, GPX4A) all increased significantly high. In addition, after ethanol treatment, we detected transcriptional activation of thioredoxin 4 (PRDX4) and thioredoxin-like 1/4 (TXNL1, TXNL4) in the thioredoxin system. The quantitative results of active oxygen show that ethanol can increase the active oxygen in the liver. In addition, GSH decreased significantly after ethanol exposure, and the ratio of GSH/GSSG increased, suggesting that oxidative stress may play an important role in the pathogenesis of adult zebrafish liver.

　　Conclusion: Under low-dose chronic ethanol exposure, adult zebrafish can reproduce the pathology, metabolism and stress response of ALD patients well. Therefore, our adult zebrafish ALD model can be used as another animal model for studying human chronic liver disease.