急性肝损伤 z-bo 2020-10-15 560

　　Abstract: Chronic alcohol abuse is common and the main cause of alcoholic liver disease (ALD). However, it is still difficult to find a safe and effective treatment for ALD. In this study, we evaluated the utility of adult zebrafish as an in vivo model in rapidly assessing the efficacy of drugs for alcohol-induced acute liver injury. We exposed adult zebrafish to 0.5% ethanol for 24, 48, and 72 hours, and measured serum alanine aminotransferase (ALT) activity. Compared with the untreated control group, ethanol treatment resulted in a significant increase in ALT levels after 48 hours and 72 hours. At the same time, during ethanol exposure, the mRNA expression of inflammation-related genes in the liver increased significantly. In order to evaluate the effectiveness of our zebrafish model for alcoholic acute liver injury drug trials, previously in rodent models of alcoholic liver disease, we studied nicotinamide riboside (NAD+ substrate) and TES-1025 (α- Amino-β-carboxymucic acid-ε-semialdehyde decarboxylase (ACMSD) inhibitor can increase the protective effect of NAD+). We found that both nicotinamide riboside and TES-1025 treatments can inhibit ethanol-induced serum ALT levels after 48 hours of exposure to ethanol. In a similar manner, riboflavin supplementation also suppressed ethanol-induced elevation of serum ALT during ethanol exposure. In addition, both nicotinamide riboside and riboflavin supplementation inhibited the up-regulation of mRNA expression of genes related to inflammation and neoadipogenesis. In summary, we have established an adult zebrafish ethanol-induced acute liver injury model, which will help in vivo drug screening, which may provide new treatments in the future to reduce liver injury related to excessive drinking.

　　Introduction: The rodent model of Alcoholic Liver Disease (ALD) has been widely used for decades. It adopts a Lieber-DeCarli liquid diet containing ethanol and is fed ad libitum. However, in rodent models, this diet can only cause mild liver steatosis even after drinking for up to 9 months. In recent years, adult zebrafish has become a more general animal model for studying ALD. We have previously demonstrated that low-dose ethanol exposure for 4 weeks can cause liver steatosis and liver damage, accompanied by mild liver fibrosis. Therefore, the zebrafish ALD model is more sensitive to ethanol exposure. However, both of these ALD models require long-term ethanol exposure, which limits the model's application in rapidly identifying therapeutic molecules to prevent liver damage. Although there are many in vivo models of acute alcohol exposure available, including primates, dogs, pigs, and rodents, these models are costly and technically challenging. In order to overcome the limitations of the past, we developed and optimized the conditions. Using adult zebrafish as experimental materials, we studied the mechanism of ethanol-induced acute liver injury, and further tested the protective effect of candidate molecules on liver cell survival, against ethanol-induced acute liver injury. liver damage.

　　Zebrafish breeding and ethanol treatment: AB / TU (wild type) zebrafish strains (more than 12 months old) were used in this study. Before the start of the test, feed once a day in the morning. Randomly select each group of wild-type zebrafish and place them in separate glass water tanks (3 fish per tank, 6 tanks in total), which contain 4 L of water, with or without 0.5% ethanol (20 ml ethanol/4 L water). Change the water with or without ethanol every 24 hours.

　　Drug treatment: Nicotinamide ribose chloride was dissolved in water (1 mg/mL), and diluted to a concentration of 16μg/L or 160μg/L in the water tanks of the control group and the ethanol group. TES-1025 was dissolved in DMSO (100 mg/mL) and treated at a concentration of 192μg/L for 2 days during ethanol exposure. Dissolve riboflavin in 0.05 N NaOH (1 mg/mL) and treat it at a concentration of 60 μg/L or 600 μg/L.

　　Blood collection and biochemical analysis: Insert a heparin-coated glass capillary needle into the dorsal aorta of zebrafish for blood collection. The blood sample was diluted 1:10 in PBS. After centrifugation at 700 g for 3 min, the supernatant was collected. Measure the alanine aminotransferase (ALT) level in the diluted serum using the ALT test kit according to the manufacturer's protocol.

　　Adult zebrafish liver damage caused by short-term high-dose ethanol exposure: To establish the best conditions for rapid evaluation of the efficacy of alcohol-induced liver injury drugs, adult zebrafish were exposed to 0.5% ethanol for 24, 48 and 72 hours, and then the changes in serum ALT levels were measured . Ethanol exposure for 24 hours can cause a moderate increase in ALT, and exposure for 48 or 72 hours can significantly increase serum ALT levels. Histological analysis of control and ethanol-exposed zebrafish liver revealed an increase in the number of swollen hepatocytes with large vacuoles. We further tested the changes in ALT levels with a larger sample size (n=9 zebrafish) and confirmed the consistency and minimal changes in ALT levels. The mRNA expression of inflammation-related genes was significantly increased, which is consistent with acute liver injury caused by alcohol exposure.

　　Short-term high-dose ethanol exposure causes liver damage in adult zebrafish. A) Expose 3 adult zebrafish to 0.5% ethanol (20ml ethanol in 4 liters of water) for 1 to 3 days, and measure serum ALT levels to determine the degree of liver damage. (B) Blood samples were collected from untreated control (white) and adult zebrafish exposed to ethanol (blue) at 24, 48, and 72 hours, and serum ALT levels were determined. (C) Representative image of HE staining in control and ethanol-exposed liver (n = 5). Scale bar = 25μm. (D) ALT levels of the three groups of control group and ethanol exposure group. (E) The relative expression of tumor necrosis factor α (tnfa), activated B cell nuclear factor-κ light chain enhancer (nfkb) and interleukin 1β (il1b)) mRNA in the liver of the control group and ethanol exposure group (n = 3) . (F) Changes in serum ALT levels after exposure to 0.5% ethanol for 48 hours.

　　Fast recovery from ethanol-induced liver injury in zebrafish: Since zebrafish show rapid regeneration after various tissue injuries, we tried to study the changes in serum ALT levels after ethanol removal. In this experiment, ethanol-treated zebrafish were transferred to a tank containing fresh water, and serum ALT levels were measured at 6, 12, and 24 hours after ethanol exposure. We found that the elevation of ALT induced by ethanol dropped to a normal level within 12 hours.

　　The inhibitory effect of nicotinamide riboside, TES-1025 and riboflavin on ethanol-induced acute liver injury: To determine the efficacy of small molecules that can protect liver cells from ethanol-induced acute liver injury, we tested nicotinamide riboside (NR) (NAD + substrate), this compound has previously been shown to inhibit ethanol-induced liver damage in rodent models. We found that NR supplementation inhibited ethanol-induced ALT elevation. We also tested TES-1025, which is an inhibitor of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), which has been shown to increase NAD+ levels. Adding TES-1025 to ethanol-treated zebrafish can increase liver NAD+ levels, indicating that both NR and TES-1025 can protect liver cells from long-term ethanol-induced liver damage. Studies have shown that long-term drinking can reduce the absorption of intestinal riboflavin. Because riboflavin is a substrate formed by flavin adenine dinucleotide (FAD), ethanol may cause a decrease in liver FAD. Since FAD is an important cofactor for many mitochondrial enzymes involved in energy metabolism, redox homeostasis and protein folding, the reduction of FAD may be one of the reasons for ethanol-induced liver damage. We tested whether riboflavin can reduce alcohol-induced liver injury and found that riboflavin supplementation can inhibit alcohol-induced liver injury. In addition, we found that NR or riboflavin-treated adult zebrafish liver damage was inhibited in a dose-dependent manner during ethanol exposure.

　　Nicotinamide riboside, TES-1025 and riboflavin supplementation during ethanol exposure can inhibit acute liver damage caused by ethanol. (A) Schematic diagram of drug testing. (B) The change of serum ALT in the control group (white bars) and ethanol exposure group (blue bars) after taking or not taking drugs. (C) (C) Dose-dependent inhibition of ALT by supplementing with NR during ethanol exposure. (D) The expression of mRNA related to inflammation and lipogenesis in the control group and ethanol-exposed zebrafish liver. (E) Riboflavin supplementation during ethanol exposure can inhibit ALT in a dose-dependent manner. (F) The control group with or without riboflavin and the expression of mRNA related to inflammation and lipogenesis in the ethanol-exposed zebrafish liver.

　　Conclusion: We have established an adult zebrafish model of ethanol-induced acute liver injury. Our findings also raise an interesting possibility that supplementing certain compounds through diet during binge eating or heavy drinking can reduce the degree of liver damage, and protecting liver cells from ethanol-induced acute liver damage can prevent alcohol The development of steatohepatitis.