| BackgroundNon-alcoholic fatty liver disease (NAFLD) is a kind of clinical syndrome, characterized by triglyceride deposit in the hepatocytes in patients who do not consume excessive ethanol. NAFLD encompasses a wide spectrum of diseases, including simple fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fibrosis and non-alcoholic cirrhosis. Generally, NAFLD is complicated with obesity and (or) diabetes mellitus type 2, dyslipidemia and its related cardiac-cerebral vascular diseases. NAFLD is also closely associated with decompensated hepatic cirrhosis, liver failure and primary hepatic carcinoma. With the development of society economy, NAFLD becomes the second reason of chronic liver diseases following viral hepatitis and it seriously hazard health and lives of people in China.The pathogenesis and drug therapy of NAFLD are the main problems that perplex us in the NAFLD researches. The underlying mechanism for the development and progression of NAFLD is complex and is still incompletely understood at present. The ’two-hit hypothesis’ proposed by Day is the leading theory for the pathogenesis of NAFLD. The ’first hit’ is defined as the accumulation of triglyceride and free fatty acid in the liver by insulin resistance. The hepatocytes with fat sensitizing the liver to the stimulus of endogenous and exogenous factors, acting as a ’second hit’. The ’second hit’ activates oxidative stress, lipid peroxidation, the releasing of cytokines and Fas ligand and results in the inflammatory and necrosis of liver cells. However, the ’two-hit hypothesis’ is not enough to disclose the whole formation process of NAFLD. Additionally, drug therapy of NAFLD is another problem that troubled us in the NAFLD researches. The backbone of therapy in patients with NAFLD lies in lifestyle modification and exterminate of incentives. The adjuvant therapies in patients with NAFLD include hepatoprotective, anti-inflammatory and anti-fibrosis drugs. However, there is no single specific pharmacologic therpy that has approved to treat patients with NAFLD in clinic at present. Therefore, it is necessary to develop the new drug for NAFLD treatment.In order to investigate the pathogenesis of NAFLD and develop potentially therapeutic drugs, it is necessary to establish an economic, simple and practical animal model for the NAFLD study. Compared to rodent models, zebrafish have many advantages:small size, large numbers of progeny and easy raising; rapid development, short latency period and shorter test period; transparent embryos, easy to observe their internal organs and disease in vivo; annotation to zebrafish genome sequence has been completed; easy to obtain sufficient sample size and suit for perform high-throughput screening. However, NAFLD model in zebrafish has not been reported yet in China. In the present study, based on the physiological and structural characteristics, zebrafish larvae were treated with high fat diet and high fat plus two different concentrations of high cholesterol diets in order to establish a NAFLD model. Based on HFC diet-induced hepatic steatosis model in larvae, further study was carried on to detect the lipid-lowing effect of ezetimibe and simvastatin and confirm the reliability of the model. Finally, the relative genes and proteins expression involved in lipid metabolism, ER stress and inflammation pathways are detected to explore the underlying mechanism of NAFLD. Our study could bring some new ideas in mechanism researches and related drug discovery of NAFLD.Aim1. To establish a simple, practical and reproducible NALFD model in zebrafish larvae induced by high fat diet and high fat plus high cholesterol diet.2. To observe the lipid-lowing effect of ezetimibe and simvastatin using NAFLD model in zebrafish larvae, including survival rate, body length and body weight, biological indexes, lipid droplets and the pathological changes in liver tissues.3. To explore the underlying mechanism of NAFLD, including the changes of relative genes and proteins expressions in lipid metabolism, ER stress and inflammation pathways.Materials and Methods1. AnimalsZebrafish (Wild-type AB strain) were offered by the key laboratory of Zebrafish model of human disease and drug screening, Southern Medical University. Zebrafish were raising according to Westerfield protocol. For modeling, Zebrafish larvae with normal development were randomly divided into four groups:Control group, high-fat group (HF), high-fat plus 2.5% high-cholesterol group (2.5% HFC) and high-fat plus 5.0% high-cholesterol group (5.0% HFC). For drug study, Zebrafish larvae with normal development were randomly divided into four groups:Control group,2.5% HFC group,2.5% HFC+ezetimibe group and 2.5% HFC+simvastatin group. Ezetimibe used in this study was added directly to the fish water at concentrations of1 μM and simvastatin was added at 50 μg/g food. These concentrations of drugs and methods of administration were chosen based on previous report as described.2. General observation of zebrafish larvaeThe conditions of growth, food-intake, activities and death rate were observed every day. The body length and body weight were measured at the time point.3. Whole-mount oil red O staining of zebrafish larvaeZebrafish larvae in each group were collected in six-well plate and fixed with 4% paraformaldehyde overnight at 4℃ and washed twice with PBS, infiltrated with 80% and 100% propylene glycol,20 minutes respectively and stained with fresh ORO overnight at room temperature in dark. Next day, sained larvae were washed twice with PBS and 100% and 80% propylene glycol followed by 30 minutes each and infiltrated with 80% glycerol at 4℃. Lipid droplets in hepatocytes were observed and imaged on an Olympus szx10 microscope and the incidence of hepatic steatosis was recorded.4. Oil red O staining of cryosections of zebrafish larvaeZebrafish larvae in each group were collected in 1.5ml EP tubes and fixed in 4% PFA overnight at 4℃. Next day, larvae were infiltrated with 30% sucrose. Larvae were embedded in Tissue-Tek OCT Compound and 10 μm sections were mounted on slides and stained with oil red O. Slides were visualized on an Olympus BX51 microscope.5. Histologic analysis and H&E staining of zebrafish larvaeZebrafish larvae in each group were collected in 1.5ml EP tubes and fixed in 4% PFA overnight at 4℃. Next day, zebrafish larvae were embedded in paraffin according to standard procedures. Four μm sections were stained with hematoxylin-eosin (H&E) and imaged on an Olympus BX51 microscope.6. Biological analysis of zebrafish larvaeThe livers of 50-70 zebrafish larvae were isolated and homogenized in lysis buffer, centrifuged and got supernatant liquid. FC, TC and TG in the liver homogenates were measured using the kits following the manufacturer’s instructions.7. Real-time RT-PCRThe livers of 20-30 zebrafish larvae were isolated and collected in 1.5ml EP RNA free tubes. RNA isolated with Trizol, the RNA concentration and purity was detected by UV spectrophotometry (Nanodrop, Thermo Fisher Scientific, USA). Reverse-transcribed with PrimeScriptTM RT reagent Kit with gDNA Eraser. Real-time quantitative PCR was carried out on Light Cycler 480 (Roche, Basel, Switzerland) using gene-specific primers.8. Western-bolttingThe livers of 20-30 zebrafish larvae were isolated and lysed in RIPA buffer. The total protein was extracted according to standard procedures. The protein concentration was detected by BCA method. The entire extraction was resolved by SDS-PAGE, transferred to a PVDF membrane and incubated overnight with antibodies at 4℃.9. Statistical analysesStatistical analysis was carried out by using SPSS 16.0 statistical software. The results are expressed as mean ± SD. One-way ANOVA is perform for analysis. Graphpad Prism 5 (Graphpad, La Jolla, CA, USA) was used to draw figures. Differences were considered significantly at P< 0.05 level.ResultsChapter one Establishment of a NAFLD model in zebrafish larvae1. Effects of HF and HFC diets on the survival rate of zebrafish larvaeThe survival rate of zebrafish larvae in control, HF,2.5%HFC and 5.0%HFC groups were 96.67±5.79%,88.66±8.59%,90.00±5.93%,94.67±5.89% after 10 days feeding. There were no significant differences among these groups (P> 0.05).2. Effects of HF and HFC diets on the body length of zebrafish larvaeBody length was significantly higher in the HF group and HFC groups compared to the control after 7 days and 10 days feeding (P<0.01,n=90), there was no significant difference between 2.5% HFC group and 5.0% HFC group (P> 0.05, n=90).3. Effects of HF and HFC diets on the body weight of zebrafish larvaeBody weight was significantly higher in the HF group and HFC groups compared to the control after 7 days and 10 days feeding (P<0.01, n=90), there was no significant difference between 2.5% HFC group and 5.0% HFC group (P> 0.05, n=90).4. HF and HFC diets induce excessive lipid in the liver of zebrafish larvaeThe results of whole-mount oil red O staining and oil red O staining of cryosections indicated that comparing with the control group, the dyeing is darker and excessive lipid droplets were observed in the livers of HF and HFC groups. Moreover, zebrafish larvae fed with HF diet developed mild steatosis, whereas zebrafish given HFC diet developed obvious steatosis.5. Effects of HF and HFC diets on the percentage of steatosis of zebrafish larvaeThe percentage of steatosis was significantly higher in the HF group and HFC groups compared to the control after 7 days and 10 days feeding (P<0.001, n> 100).6. Effects of HF and HFC diets on the biological indexes of zebrafish larvaeThe level of TG, TC and FC significantly higher in the livers of zebrafish larvae in HF group and HFC groups compared to the control after 7 days and 10 days feeding (P<0.05). Comparing with HF group, the level of TC and FC significantly higher in the livers of zebrafish larvae in HFC groups (P<0.001).7. Effects of HF and HFC diets on the liver pathological features of larvaeHF and HFC groups showed structural abnormalities of liver cells and lipid droplet-induced nucleus displacement, which was not found in control group.Chapter Two Effects of ezetimibe and simvastatin on HFC diet-induced NAFLD1. Effects of ezetimibe and simvastatin on the survival rate of zebrafish larvaeThe survival rate of zebrafish larvae in control,2.5%HFC,2.5%HFC+ezetimibe and 2.5%HFC+simvastatin groups were 85.00±5.22%,80.00±4.20%,82.67±5.47%, 81.17±5.12% after 10 days feeding. There were no significant differences among these groups (P> 0.05).2. Effects of ezetimibe and simvastatin on the body length of zebrafish larvaeCompared with control group, the body length in 2.5% HFC group had increased significantly (P=0.000, n=90). Compared with 2.5% HFC group, the body length had reduced in 2.5%HFC+ezetimibe group and 2.5%HFC+simvastatin group, with statistical significance (P<0.01, n=90) after 7 days and 10 days feeding.3. Effects of ezetimibe and simvastatin on the body weight of zebrafish larvaeCompared with control group, the body weight in 2.5% HFC group had increased significantly (P=0.000, n=90) after 7 and 10 days feeding. Compared with 2.5% HFC group, the body length had reduced in 2.5%HFC+ezetimibe group and 2.5%HFC+simvastatin group, with statistical significance (2.5%HFC+ezetimibe group vs.2.5% HFC group, P=0.011; 2.5%HFC+simvastatin group vs.2.5% HFC group,P=0.001) after 7 days feeding. Compared with 2.5% HFC group, the body length had reduced in 2.5%HFC+ezetimibe group and 2.5%HFC+simvastatin group, with no statistical significance (2.5%HFC+ezetimibe group vs.2.5% HFC group, P=0.291; 2.5%HFC+simvastatin group vs.2.5% HFC group, P=0.127) after 10 days feeding.4. Effects of ezetimibe and simvastatin on lipid deposition in the livers of larvaeThe results of whole-mount oil red O staining and oil red O staining of cryosections indicated that deeper dyeing and greater lipid droplets were observed in the livers of 2.5% HFC group compared to control group. Compared with 2.5% HFC group, lipid droplets were decreased in the livers of 2.5% HFC+ezetimibe and 2.5% HFC+simavastatin groups. Moreover, ezetimibe had better lipid-lowing effect compared to simvastatin.5. Effects of ezetimibe and simvastatin on biological indexes in the livers of larvaeThe level of TG, TC and FC significantly higher in the livers of zebrafish larvae in 2.5% HFC group compared to the control group (P<0.001). Compared with 2.5% HFC group, the level of TG, TC and FC were decreased in the livers of 2.5% HFC+ezetimibe and 2.5% HFC+simavastatin groups (2.5%HFC+ezetimibe group vs. 2.5% HFC group, P<0.01; 2.5%HFC+simvastatin group vs.2.5% HFC group, P<0.05). Compared with 2.5% HFC+simavastatin group, the level of TC and FC were lower in the livers of 2.5% HFC+ezetimibe group (P<0.001).6. Effects of ezetimibe and simvastatin on liver pathological features of larvaeThe structures of hepatocytes in control group are normal, without excessive lipid droplets. However, the structure of hepatocytes in 2.5% HFC group was abnormal and diffuse lipid droplets were found 2.5% HFC group. Compared with 2.5% HFC group, fewer lipid vacuoles were found in 2.5% HFC+ezetimibe and 2.5% HFC+simavastatin groups.Chapter Three Study on the pathogenesis of NAFLD and the underlying mechanism of ezetimibe and simvastatinPart one Study on the pathogenesis of NAFLD1. Effects of HF and HFC diets on the genes expression involved in lipid metabolism in the livers of zebrafish larvaeAfter 10 days feeding, the relative mRNA expressions of fasn and hmgcra were reduced in HF group compared with the control group (P<0.05), whereas the relative expressions of pparab, cptla and acox3 were significantly higher compared with the control group (P<0.01). Moreover, the relative mRNA expressions of acaca, fasn, srebf2, hmgcsl and hmgcra were reduced in HFC groups compared with the control group (P<0.05), whereas the relative expressions of pparab, cpt1α and acox3 were significantly higher compared with the control group (P<0.05).2. Effects of HF and HFC diets on the genes expression involved in ER stress in the livers of zebrafish larvaeAfter 10 days feeding, the relative mRNA expressions of atf6, ern2, hspa5 and hsp90bl were increased in HF group compared with the control group (P=0.000, P<0.05), whereas the relative mRNA expressions of eif2ak3 and ddit3 had no significant difference between two groups (P> 0.05). Moreover, the relative mRNA expressions of atf6, hspa5 and hsp90bl were higher in HFC groups compared with the control group (P<0.001). After 20 days feeding, the relative mRNA expressions of atf6, hspa5, hsp90bl and ddit3 were increased in HF and HFC groups compared with the control group (P<0.05).3. Effects of HF and HFC diets on the genes expression involved in inflammation pathway in the livers of zebrafish larvaeAfter 10 days feeding, the relative mRNA expressions of il6 was increased in 5.0% HFC group compared with the control group (P=0.005), whereas the relative mRNA expressions of tnfa and illb had no significant difference among groups (P> 0.05). After 20 days feeding, the relative mRNA expressions of tnfa, illb and il6 were increased in HF group compared with the control group (P<0.05). Moreover, the relative mRNA expressions of tnfa, illb and il6 were increased in HFC groups compared with the HF group.Part two Study on the underlying mechanism of ezetimibe and simvastatin1. Effects of ezetimibe and simvastatin on the genes expression of srebf2, hmgcsl and hmgcra in the livers of zebrafish larvaeThe relative mRNA expressions of srebf2, hmgcsl and hmgcra were reduced in 2.5% HFC group compared with the control group, with statistical significance (P<0.01). There were no statistical differences in the expression levels of srebf2, hmgcsl and hmgcra mRNA between 2.5% HFC+ezetimibe and control group (P> 0.05). The relative mRNA expressions of srebf2, hmgcsl and hmgcra were also reduced in 2.5% HFC+simvastatin group compared with the control group, with statistical significance (P<0.01).2. Effects of ezetimibe and simvastatin on the genes expression of pparab, cpt1α and acox3 in the livers of zebrafish larvaeThe relative mRNA expressions of pparab, cpt1α and acox3 were upregulated in 2.5% HFC group compared with the control group, with statistical significance (P<0.01). There were no statistical differences in the expression levels of pparab, cpt1α and acox3 mRNA between 2.5% HFC+ezetimibe and 2.5% HFC group (P> 0.05). Compared with 2.5% HFC group, the expression level of acox3 mRNA in 2.5% HFC+simvastatin group decreased, with statistical significance (P=0.018), whereas there were no statistical differences in the expression levels of pparab and cpt1｛ mRNA between 2.5% HFC and 2.5% HFC+simvastatin group (P> 0.05).3. Effects of ezetimibe and simvastatin on the genes expression of atf6, hspa5 and hsp90b1 in the livers of zebrafish larvaeThe relative mRNA expressions of atf6, hspa5 and hsp90bl were upregulated in 2.5% HFC group compared with the control group, with statistical significance (P<0.001). Compared with 2.5% HFC group, the expression level of atf6, hspa5 and hsp90b mRNA in 2.5% HFC+ezetimibe group decreased, with statistical significance (P<0.05). Compared with 2.5% HFC group, the expression levels of atf6, hspa5 and hsp90b mRNA were reduced in 2.5% HFC+simvastatin group, with statistical significance (P<0.05).4. Effects of ezetimibe and simvastatin on the protein expression of GRP78/BiP in the livers of zebrafish larvaeThe relative protein expression of GRP78/BiP were upregulated in 2.5% HFC group compared with the control group. Compared with 2.5% HFC group, the relative protein expression of GRP78/BiP was reduced in 2.5% HFC+ezetimibe and 2.5% HFC+simvastatin group.Conclusions1. A model of non-alcoholic fatty liver disease was successfully established in zebrafish larvae treated with high-fat and high-fat plus high-cholesterol diet. This model may represent a useful tool for testing potentially therapeutic drugs and investigating the NAFLD pathogenesis.2. Ezetimibe and simvastatin treatment ameliorate hepatic steatosis in HFC diet-fed zebrafish larvae. Ezetimibe and simvastatin may regulate the key genes involved in the process of lipid metabolism and ER stress.3. Zebrafish can be used as an effective preclinical model organism to study NAFLD and perform high-thoughput screening of drugs in vivo. Our study could bring some new ideas in drug discovery of NAFLD. |
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