Antarctic Krill Oil Ameliorates Liver Injury In Rats Exposed To Alcohol By Regulating Bile Acids Metabolism And Gut Microbiota

Objective Antarctic Krill Oil（AKO）is rich in phospholipid,eicosapentaenoic acid（EPA）,docosahexaenoic acid（DHA）and astaxanthin,which has antioxidant and anti-inflammatory activities can regulate lipid and glucose metabolism,and has a certain protective effect on liver injury.In this study,a rat model of alcoholic liver injury was established to investigate the protective effect of AKO on alcoholic liver injury by regulating bile acids（BAs）metabolism and intestinal flora.Methods1.Animals and Experimental Design:Seventy-five male Sprague-Dawley（SD）rats（age,8weeks）,weighing 180～220 g,were divided randomly into five groups with 15 rats in each group after acclimation for one week:NC,normal control group,normal diet gavage with soybean oil（0.1 m L/d）;AC,alcohol control group,normal diet gavage with alcohol（56%（v/v）alcohol 8 m L/kg/d 4 w+10 m L/kg/d 12 w）;LAKO,low-dose AKO-treatment group,normal diet gavage with AKO and alcohol（AKO 100 mg/kg/d,alcohol dose was the same as model group）;HAKO,high-dose AKO-treatment group,normal diet gavage with AKO and alcohol（AKO 200 mg/kg/d,alcohol dose was the same as the model group）;AKOC,AKO control group,normal diet gavage with AKO（AKO 200 mg/kg/d）.Food intake was recorded daily,body weight was measured weekly,and feces were collected.After 16weeks of treatment,the rats were sacrificed by pentobarbital(40 mg·k^-1,i.p.).Blood samples,liver,and intestinal tissues were rapidly collected.A portion of liver tissues was fixed.The remaining tissues were stored at-80°C for further examination.2.Liver pathological examination:Hematoxylin-Eosin（H&E）staining was used to observe the pathological changes of liver tissue,transmission electron microscope was used to observe the ultrastructural changes of rat liver cells.3.Biochemical Analysis:Automatic Biochemical Analyzer（Beckman Coulter,America）was used to measure aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,γ-glutamyl transpeptidase（GGT）,cholinesterase level,triacylglycerol（TG）,total cholesterol（TC）,high-density lipoprotein cholesterol（HDL-C）and low-density lipoprotein cholesterol（LDL-C）.4.Samples of blood,liver,and feces for BAs profiling and Western blot:High Performance Liquid chromatography-mass Spectrometry/Mass Spectrometry（LC-MS/MS）were used to quantitatively determine the BAs in the liver,serum and feces of rats.Western blot（WB）was used to detect the levels of farnesoid X receptor（FXR）,cholesterol 7α-hydroxylase in the liver and ileum of rats（CYP7A1）,sterol 12α-hydroxylase（CYP8B1）,fibroblast growth factor receptor 4（FGFR4）and Fibroblast growth factor 15（FGF15）BAs-related proteins.5.DNA Extraction and 16S r DNA Sequencing:16S r DNA high-throughput sequencing technology was used to analyze the differences of intestinal microflora among all groups.Results1.The results of body weight,food intake,energy and liver index showed that body weight and food intake of the AC group were significantly decreased compared with the NC group（P<0.05）.Compared with the AC group,the body weight of the HAKO group was significantly increased（P<0.05）.There was no significant difference in energy intake among all groups（P>0.05）.The liver index of the AC group was significantly lower than that of the NC group（P<0.05）.The liver index in the HAKO group was significantly higher than that in the AC group（P<0.05）.2.H&E staining of the liver sections showed that the hepatocytes had structural intactness without lipid droplets in the NC group and AKOC group.However,hepatic cord derangement,inflammatory infiltration,hepatocytes severe swelling,and fat vacuoles diffusing were obviously increased in the AC group.Rats in the LAOK group and the HAKO group showed significant improvement of histopathological changes.3.In the NC and AKOC groups,the hepatocytes were structurally intact without lipid droplets and the bile duct was pure following morphologically normal microvilli of timid tubes.Mitochondria and endoplasmic reticulum were maintained intact structures with clearly visible mitochondrial cristae and orderly arrangement of endoplasmic reticulum.In the AC group,there were a large number of lipid droplets,broken endoplasmic reticulum,disordered arrangement,mitochondrial swelling,fuzzy mitochondrial crest,abnormal microvilli of the ciliary duct,and foreign bodies in the bile duct.However,alcohol exposure led to fractured endoplasmic reticulum,swollen and deformed mitochondria with vague mitochondrial cristae,increasing droplet fat and abnormal microvilli of timid tubes.The bile duct abounded with foreign matter.In the HAKO group,the damage of ultrastructure was strikingly improved.4.Serum biochemical indices showed that ALT,AST and GGT levels in the AC group were significantly increased,compared with the NC group（P<0.05）.Compared with the AC group and LAKO group,serum ALT,AST and GGT levels of the HAKO group and AKOC group were significantly decreased（P<0.05）.In addition,the serum TG,TC and LDLC levels in the AC group and LAKO group were significantly higher than those in the NC group（P<0.05）.The serum TG,TC and LDLC levels in the HAKO group and AKOC group were significantly lower than those in the AC group（P<0.05）.5.The results of serum BAs level showed that the levels of glycine-conjugated BAs in serum and liver of the AC group were significantly decreased,and taurine conjugated BAs in serum was significantly decreased,compared with the NC group（P<0.05）.Compared with the AC group,serum levels of total BAs,unconjugated BAs and glycine-conjugated BAs in the HAKO group were significantly decreased（P<0.05）.During the intervention period,the AC group showed a decreasing trend in fecal unconjugated BAs,glycine-conjugated BAs and taurine-conjugated BAs,while the HAKO group showed a decreasing trend in significantly improved BAs levels（P<0.05）.6.WB results showed that compared with the NC group,hepatic FXR and FGFR14expression levels in the AC group were significantly decreased by 56.1%and 54.9%,respectively（P<0.05）.And the expression levels of FXR and FGF15 in the ileum were significantly decreased by 61.6%and 54.5%,respectively,and the expression levels of CYP7A1 and CYP8B1 in the liver were significantly increased by 48.4%（P<0.05）.The HAKO group significantly improved alcohol-induced bile acid reduction.7.16S r DNA sequencing results showed that the abundance of Intestinimonas and Methanobrevibacter in the AC group was significantly increased（P<0.05）.The abundance of Methanobrevibacter and Psychrobacter in high dose group decreased significantly（P<0.05）.Conclusion1.Proper supplementation of AKO can effectively improve liver injury in rats exposed to alcohol.2.Appropriate supplementation of AKO can effectively improve alcoholic liver injury.The possible mechanism is that AKO regulates the disorder of BAs metabolism and may improve the diversity and structural changes of intestinal flora.