To Explore The Effect Of Naringenin In Alcoholic Fatty Liver In Zebrafish And Establish Caveolin-1 Gene Zebrafish Mutant

Background:Alcoholic liver disease(ALD)is a spectrum of hepatic abnormalities that extend from isolated alcoholic steatosis to steatohepatitis and cirrhosis.Studies have shown that naringenin can remove free radicals and protect the normal morphology and function of nucleic acid,protein and fat.Naringenin also has strong biological activity in reducing blood fat and improving lipid metabolism.Naringenin can improve fat metabolism,but there is no related research to study the effect of naringenin in alcoholic fatty liver.Compared to traditional alcohol fatty liver model in mice,zebrafish model has obvious advantage because of simple operation,less time-consuming,easy observation,less dosage of samples and less drugs required in experiment.Caveolin-1(Cav-1)plays an important role in cholesterol transport and lipid metabolism,and it participates in the development of liver diseases.CRISPR/Cas9 system is the most commonly used gene editing technique by now,and zebrafish mutants have been widely used in experimental study.But there is no research studied liver diseases by establishing cav-1 gene zebrafish mutant.Objective:To explore the effect of naringenin and cav-1 in alcoholic fatty liver in zebrafish and establish cav-1 gene zebrafish mutants.Methods:To microinject cas9 mRNA and cav-1 guide RNA to one-celled zebrafish embryos and establish cav-1 gene zebrafish mutants by CRISPR/Cas9.To establish an acute alcoholic fatty liver model by exposing 4 days post fertilization(dpf)zebrafish larvae to 2%ethanol for 32 hours and intervene by adding different concentrations of naringenin.To assess the model and the therapeutic effect of naringenin through whole oil red O staining and H&E staining of paraffin section.To explore the preliminary mechanism of therapeutic effect through TUNEL staining of paraffin section,as well as measuring the expression of cav-1 mRNA and protein by Real-time Quantitative PCR and Western Blot.Results:We completed cutting targeted sequences of cav-1 gene in zebrafish and formed cav-1 gene zebrafish mutants.They have transmissibility.After 32 hours of exposure to 2%ethanol,larvae were observed serious lipid accumulation in liver,which was detected by whole oil red O staining and paraffin section H&E staining.Naringenin significantly down-regulated hepatic steatosis with a dose-dependent change and the dosages(5mg/L and 10mg/L)almost reverse the alcoholic lipid accumulation in larvae.Naringenin significantly reduced the expressions of cav-1 mRNA and protein in zebrafish larvae.Furthermore,naringenin also attenuated hepatic apoptosis in larvae detected by TUNEL staining.Conclusion:We established cav-1 gene zebrafish mutants and they have transmissibility.Alcoholic fatty liver model in zebrafish larvae was established successfully.Naringenin inhibited alcohol-induced liver steatosis and injury in zebrafish larvae by regulating cav-1 gene and reducing apoptosis.