| Background&AimBinge drinking becomes a hot issue in current society, either acute alcohol exposure or chronic alcohol abuse can lead to severe liver injury. Alcoholic fatty liver (AFL) is the earliest and the most common form of liver injury which often caused by alcohol abuse. It can progress to alcoholic hepatitis, alcoholic fibrosis, alcoholic cirrhosis or even hepatic carcinoma. Surveys indicate that, fatty liver developed in more than90%heavy drinkers,20-40%of AFL patients suffer from advanced fibrosis while8%-20%develop liver cirrhosis and3%-10%of them progress to hepatocellular carcinoma. AFL becomes the second reason of hepatic injury following viral hepatitis in China and it becomes a heavy health burden at present.However, the pathogenesis of AFL is still incompletely understood at present, available target therapies are still lack. It is necessary for us to establish a simple and practicable animal model in order to investigate the pathogenesis of AFL and find out potentially preventive and therapeutic drugs. In the traditional way, rats and mice are using to establish models of AFL in China and aboard and it usually takes about1-16weeks to set up these models. Intragastric feeding and introperitoneal injection are using to help rodent to obtain alcohol. Nevertheless, these models are hard to operate, time-consuming and costly. What’s more, the sample size is limited. Among vertebrates, Zebrafish have emerged as a powerful system in recent years. Compared to rodent models, Zebrafish have many advantages:low cost, easy raising; rapid development and large numbers of progeny, easy to obtain sufficient sample size; in vitro fertilization, transparent embryos, easy to observe their liver; small size, take up less space, easy to perform high-throughput screening. Further study has indicated that Zebrafish can be an ideal model organism to study alcoholic liver diseases and perform high-thoughput screening of drugs for related liver diseases in vivo because they possess the pathways to metabolize alcohol, their liver is mature by4days post-fertilization and alcohol can be simply added to their water. However, the alcohol-induced acute fatty liver model in Zebrafish has not been reported yet in China.Magnesium isoglucyrrhizinate (MgIG) is a single stereoisomer of glycyrrhizic acid. MgIG, as the fourth generation of glycyrrhizic acid agents, has stronger effects, such as anti-inflammatory, protection of liver cell membrane and regulation of liver function. In clinical practice, it is mainly used in the treatment of patients with chronic viral hepatitis and has a positive effect on liver function. Other basic researches also indicate that MgIG can protect chemical and drug induced hepatic injuries and non-alcoholic fatty liver disease in rodent models. Moreover, MgIG may play a positive role in alcoholic liver disease. However, its role in acute alcoholic fatty liver disease is rarely reported and its related mechanism is unclear.In the present study, based on the physiological and structural characteristics, Zebrafish were used as a model organism to establish an alcohol-induced fatty liver model. Zebrafish were treated with three different concentrations of alcohol while alcohol can simply added to their water. Survival rate, physical activity and phenotype were observed during alcohol exposure. Whole-mount Oil Red O was used to determine the liver morphology, lipid droplets in liver tissues and the percentage of fatty liver. The pathological changes were assessed by Hematoxylin-Eosin staining and the relative gene expression was detected by Real-time RT-PCR. The optimal alcohol concentration and time point were chosen to set up this model. Based on fatty liver model induced by alcohol in Zebrafish, further study was carried on to detect the hepatoprotective effect of MgIG on acute fatty liver disease and its related molecular mechanism. Our study could bring some new ideas or new ways of practice in drug discovery and related mechanism researches of alcoholic fatty liver disease.Materials and Methods1、AnimalsZebrafish (Wild-type AB strain) were provided by Laboratory of Zebrafish, Department of Cell Biology, Southern Medical University. Zebrafish were maintained according to standard procedures (Westerfield,1995).14:10hours light:dark cycle at28.5℃. Fertilized embryos collected following natural spawning were cultured at28.5℃in Holt buffer.2、Ethanol TreatmentAt5dpf (days post fertilization, dpf), larvae with normal liver development were treated with0%,2.0%,2.5%,3.0%alcohol respectively and cultured in sealed plate for up to32h in order to prevent alcohol volatilization.3、Oil Red O StainingWhole larvae were collected in staining dish and fixed with4% paraformaldehyde, washed with PBS, infiltrated with increasing concentrations of propylene glycol and stained with0.5%Oil Red O in100%propylene glycol overnight. Stained larvae were washed with decreasing concentrations of propylene glycol followed by several rinses with PBS and transferred to80%glycerol. Lipid droplets in liver tissue were observed and imaged on an Olympus szx10microscope.4、Histologic AnalysisLarvae were fixed with4%paraformaldehyde overnight, embedded in paraffin according to standard procedures.4μm sections were stained with hematoxylin and eosin (HE) and imaged on an Olympus BX51microscope.5、Reverse Transcription and Quantitative PCRA total of36fish were pooled for each group and collected in1.5ml EP tube. RNA isolated with Trizol (Invitrigen, USA), the RNA concentration and purity was detected by UV spectrophotometry (Nanodrop, Thermo Fisher Scientific, USA). Reverse transcription used PrimeScriptTM RT reagent Kit with gDNA Eraser (TAKARA). Real-time quantitative PCR was carried out on Applied Bio systems7500Real-Time PCR System.6、Statistical analysesStatistical analysis was carried out by using SPSS16.0statistical software. The results are expressed as mean±SD. T-test and LSD test are perform for analysis. Sigmaplot10.0software was used to draw figures. Differences were considered significantly at P<0.05level.ResultsChapter One Establishment of an alcohol-induced acute fatty liver model in Zebrafish1、Effects of alcohol on Zebrafish survival Starting at24h of alcohol exposure, the survival rates showed a decreasing trend that corresponded to increased alcohol concentration, so that by32h of exposure there were remarkable differences among the three alcohol treated groups:2%alcohol:100%survival,2.5%alcohol:(91.21±1.61)%survival and3%alcohol:0%survival.2、Effects of alcohol on Zebrafish phenotype and activityAbnormalities in Zebrafish phenotype and physical activity were observed at the24h time point and showed concentration-dependence.2.0%alcohol-treated group: normal morphology, increased activity, abnormal circling.2.5%alcohol-treated group: some deformities and3.0%alcohol-treated group:spinal curvature, pericardial edema, cavity hemorrhage, yolk sac edema, failure to inflate the swim bladder.3、Effects of alcohol on Zebrafish liver morphologyThe results of oil red staining indicated that the livers of Zebrafish in2.0%alcohol-treated group are obviously swelling comparing with control group after32h exposure.4、Alcohol induce excess lipid droplets in Zebrafish liver tissueComparing with control group, the intrahepatic dyeing is deepened and excess lipid droplets were found in Zebrafish liver tissue of2.0%alcohol-treated group according to the whole-mount oil red staining.5、Effects of alcohol on the percentage of fatty liver in ZebrafishSignificant fatty liver was observed in the2.0%alcohol-treated group at32h of exposure,, as evidenced by the percentage of fatty liver ((71,25±0.15)%vs. controls:(31.25±0.05)%,t=5.102, P=0.002). But not at24h((17.50±0.15)%vs. controls:(7.50±0.05)%,t=1.265, P=0.253)。6、Effects of alcohol on the pathological features of liver of ZebrafishCompared with the control group, pathological features of liver of2.0% alcohol-treated group showed that swollen size, lipid droplet-induced nucleus displacement.7、Effects of alcohol on the gene expression that related with lipid metabolismThe relative gene expression was analyzed by Real-time RT-PCR in whole larvae treated for24or32h with2.0%alcohol.24h, the expression of accl, fasn, hmgcs and hmgcra mRNA expression in2.0%alcohol-treated group were (2.9401±0.4845),(2.8733±0.7876),(6.5050±4.8122) and (3.1762±1.3638) times higher than that of control group respectively, with statistical significance (P<0.05).32h, the expression of accl, fasn, hmgcs and hmgcra mRNA expression in2.0%alcohol-treated group were(4.4459±1.1233),(3.7219±0.9065),(4.1729±1.1271) and (3.9238±0.8222) times higher than that of control group respectively, with statistical significance (P<0.05).Chapter Two Hepatoprotective effect of MgIG on alcohol-induced acute fatty liver in Zebrafish1、Effects of MgIG on liver morphology in Zebrafish with AFLThe results of oil red staining indicated that the livers of Zebrafish in model group, comparing with control group, are obviously swelling. In addition, the intrahepatic dyeing is deepened and excess lipid droplets were found in the livers of model group. The morphology of livers is normal when Zebrafish were only received MgIG (0.1mg/ml). Compared with model group, the swelling degree of the livers is lessened in MgIG (0.1mg/ml) plus2.0%alcohol-treated group.2、Effects of MgIG on the percentage of fatty liver in Zebrafish with AFLThe results of oil red staining indicated that compared with control group, the percentage of fatty liver in model group had increased significantly ((63.33±0.06)%vs.(36.67±0.08)%,P=0.002). Compared with model group, the percentage of fatty liver in Zebrafish had reduced in MgIG (0.1mg/ml) plus2.0%alcohol-treated group, with statistical significance ((37.33±0.11)%vs.(63.33±0.06)%, P=0.022).While there was no statistical differences between model group and MgIG (1mg/ml) plus2.0%alcohol-treated group ((65.00±0.15)%vs.(63.33±0.06)%, P=0.861).There was also no statistical differences between model group and MgIG (0.01mg/ml) plus2.0%alcohol-treated group ((68.33±0.12)%vs.(63.33±0.06)%,P=0.602). Thus it can be seen that MgIG (0.1mg/ml) can reduced the percentage of fatty liver in Zebrafish with AFL. Neither the concentration of MgIG (1mg/ml) nor MgIG (0.01mg/ml) would make the protective effect work.3、Effects of MgIG on the pathological features of liver of Zebrafish with AFLHE staining results showed that, the structure of hepatocyte of Zebrafish in control group is normal, without lipid vacuoles. However, the structure of hepatocyte of Zebrafish in model group was abnormal, showed as swelling, diffuse lipid vacuoles and the cytoplasm filled with small lipid vesicles. The structure of hepatocyte of Zebrafish was normal in the group that is only giving a moderate concentration of MgIG (0.1mg/ml).Compared with model group, alleviative fatty liver in Zebrafish of MgIG (0.1mg/ml) plus2.0%alcohol-treated group.Chapter Three Study on mechanism of hepatoprotective effect of MgIG on alcoholic fatty liver in Zebrafish1、Effects of MgIG on the expression of accl,fasn, hmgcs and hmgcra mRNA levels in ZebrafishThe relative gene expression was analyzed by Real-time RT-PCR in whole larvae treated with0%or2.0%alcohol, MgIG (0.1mg/ml) and MgIG (0.1mg/ml) plus2.0%alcohol. The expression of accl, fasn, hmgcs and hmgcra was (3.6372±1.4115),(3.3860±0.8487),(2.7270±1.3024) and (2.6209±2.1109) times higher in model group compared with the control group and was significantly reduced in MgIG(0.lmg/ml) plus2.0%alcohol-treated group compared with the2.0%alcohol-treated group.*P<0.05.2、Effects of MgIG on the expression of echs, mpt mRNA levels in ZebrafishThe relative gene expression was analyzed by Real-time RT-PCR. The results showed that the difference, however, was not statistically significant on the expression levels of echs mRNA among the four groups (control group, model group, MgIG (0.1mg/ml) group and MgIG (0.1mg/ml) plus2.0%alcohol-treated group).①Compared with control group, the expression level of echs mRNA in model group decreased, with no statistical significance (P=0.102). There were no statistical differences in the expression level of echs mRNA between MgIG (0.1mg/ml) plus2.0%alcohol-treated group and model group (P=0.445).②Compared with control group, the expression level of mpt mRNA in model group increased, with statistical significance (P=0.018). Compared with model group, the expression level of mpt mRNA in MgIG (0.1mg/ml) plus2.0%alcohol-treated group decreased, with statistical significance (P=0.002).Conclusions1、A model of acute alcohol-induced fatty liver was successfully established in5dpf Zebrafish treated with2.0%alcohol for32h. This model may represent a useful tool for investigating the disease pathogenesis and performing high-throughput screening of potentially therapeutic drugs.2、MgIG(0.1mg/ml)have hepatoprotective effect on fatty liver in Zebrafish. However, MgIG(1mg/ml) and MgIG (0.01mg/ml)have no hepatoprotective effect on fatty liver in Zebrafish. MgIG (0.1mg/ml) may regulate the key genes in the process of lipid metabolism and ameliorate the disorder of lipid metabolism and then protect the liver. 3、Zebrafish can be used as an effective preclinical model organism to study liver diseases and perform high-thoughput screening of drugs in vivo. The experimental results showed that MgIG might have a therapeutic effect on alcohol-mediated liver damage. |
PDF Full Text Request