Therapeutic Effect And Mechanism Of Hesperidin On Alcohol Fatty Liver In Zebrafish

Background and AimAlcoholic fatty liver disease(AFLD)is characterized by excessive accumuLation of fat in hepatocytes due to long-term alcohol abuse.It is an early stage of alcoholic liver disease(ALD),and can be effectively controlled or even reversed by timely treatment,which has great significance of the prevention and treatment of ALD.Traditional Chinese medicine has a long-term application on the treatment of AFLD and it is seems that the effect is remarkable.Therefore,to study the effect and mechanism of traditional Chinese medicine and its components on AFLD is of great significance.Hesperidin is a main constituent of tangerine peel and citrus aurantium,is proved to play a role in anti-oxidation,anti-inflammation,antihypertensive,cholesterol-lowering and many other pharmacological effects in various animal experiments.Studies have shown that it has certain effect on the therapy of AFLD.However,the underlying mechanism of resistance to alcoholic liver injury is still unclear.Zebrafish has been widely used on model establishment and drug screening because of its unique biological advantages nowadays.It has been reported that the acute alcoholic fatty liver model on zebrafish has been successfuLly established,which provide a new way to further study the pathogenesis of AFLD and carry out related drug research.Thus,we aimed to investigate the protective effects of Hesperidin against AFLD and its molecuLar mechanism in this study,based on an alcohol-induced acute fatty liver model in zebrafish.We suggest our study couLd provide some new ideas for the application and further study of traditional Chinese medicine on alcoholic fatty liver disease.Materials and Methods1.AnimalsWide-type AB strain zebrafish and transgenic line with liver specific eGFP expression Tg(lfabp10α:eGFP)zebrafish.2.Ethanol and Hesperidin Treatment4 dpf(days post fertilization)zebrafish were first randomly divided into two groups,a control group treated with fish water only and a model group exposed to 350mM ethanol for 32h.Part larvae were collected for further research.Subsequently,the model group were randomLy assigned into several groups as followed equally:model group,0.1%DMSO group,3 Hesperidin treated groups(25μg/mL,12.5μg/mL,6.25μg/mL).Hesperidin monomer was dissolved in DMSO(working solution was diluted in fish water and DMSO’s final concentration less than 0.1%).After incubated for 48h,larvae were collected for further detections.3.Experimental TechniquesThe morphological changes,lipid deposition and histopathological changes of the zebrafish liver were observed by oil red O staining,Nile red staining and HE staining.The mRNA expression of the target genes were detected by Quantitative real-time PCR.4.Statistical analysisAll data are presented as mean ± standard deviation(SD).Statistical analysis was carried by SPSS(version20).Statistical differences were evaluated by student’st test and one-way ANOVA test.Value of P<0.05 was considered to be statistically significant.Graphpad Prism 5 software was used to plot graph.Result1.We found that compared with the control group,hepatomegaly were appeared in most of the larvae and severe lipid deposited in the liver tissues after 32 hours of exposure to 350mM ethanol,so the acute alcohol fatty liver model in zebrafish is successfuLly established.2.Oil red O staining and Nile Red staining show that hesperidin could dose-dependently alleviate hepatic lipid droplets and morphological damage of liver in larvae induced by alcohol.Furthermore,paraffin sections of larvae stained with H&E also confirmed the liver pathological changes consistently.The results revealed that hesperidin could reduce the development of hepatic steatosis in zebrafish induced by alcohol.3.Hesperidin dramatically reduced and the expressions of alcohol and lipid metabolism-related genes,including cyp2y3,cyp3a65,hmgcra,hmgcrb,fasn and fads2 compared with ALD model.Moreover,the findings demonstrated that hesperidin alleviated hepatic damages as well,which is reflected by the expressions of endoplasmic reticuLum stress and DNA damages related genes(chop,gadd45aa and edeml).