The Reparation For NAFLD From Cod Skin Collagen Peptide

Non-alcoholic fatty liver disease(NAFLD)is a clinicopathologic syndrome that caused by excessive deposition of fat in liver cells,excluding drinking and other definite liver damaging factors.Currently,there is no definite and effective treatment method for NAFLD.Although pioglitazone and vitamin E have been recognized by the international community,some side effects still exist.Marine bioactive peptides are characterized by unique functions and novel structures.The development of bioactive peptides from marine biological resources has been a hot research topic in recent years.Gadus,one of the largest fish catches in the world,is often used to make fillets of cod,the skin of which is discarded as by-products.Studies have found that cod skin is rich in collagen,and the extracted collagen active peptide has been applied in many fields,but whether it has a repair effect on NAFLD has not been reported yet.So,cod skin collagen peptide(CSCP)was prepared in this study and its repair function toward to NAFND cell model and the animal models in mice.Our studies provide the experimental basis for the development of a new type of liver care products with healing effects on NAFLD.In this study,Chang liver cells were induced by oleic acid,and the in vitro NAFLD cell model was established by MTT assay for Chang liver cell activity,inverted microscope observation,oil red O staining,and gpo-pap enzyme assay for TG content in cells.The TG content in NAFLD cells was used as the standard to screen the optimal enzyme species of cod skin,and the optimal enzymatic hydrolysis process was determined by single factor and orthogonal experiment.The highest active CSCP was prepared by ultrafiltration to explore its antioxidant capacity in vitro.The obtained CSCP acted on the NAFLD cell model,and the changes of AST,ALT,MDA,SOD,GSH,CAT and ROS contents in the medium of the normal group,the model group,the low-dose CSCP group(40 ?g/mL)and the high-dose CSCP group(80 ?g/mL)were measured,respectively.Transmission electron microscope(TEM)was used to observe the ultrastructure of cells to evaluate the repair effect of CSCP on NAFLD cell model.Mice were used as experimental animals and divided into the normal group,model group,lovastatin positive control group(8 mg/kg/d),CSCP low-dose group(200 mg/kg/d)and CSCP high-dose group(400 mg/kg/d).In addition to the normal group,all other groups were given oral administration of NAFLD mice on high-fat diet.After the experiment,serum and liver were taken to calculate the liver weight index.The contents of AST,ALT,HLD,DLD,TG and SOD in the serum of mice were measured.HE staining and TEM were used to determine the changes in the tissue structure of the mouse liver.Experimental results:1.The results of MTT assay showed that Chang liver cells had the highest survival rate when the oleic acid concentration was 0.25 mM,and the vacuoles in the cells were significantly increased under inverted microscope.The results of oil red O staining showed that the lipid droplets in the cells were obviously fused and the growth morphology of the cells was good.TG content in the cells was also significantly increased,indicating the successful establishment of the NAFLD cell model.2.The best enzymolysis technology for preparation of CSCP: alkaline protease was the optimal enzyme,and solid-liquid ratio was 1:2,pH value was 9,the enzyme dosage was 2000 u/g,temperature was 50oC,and the hydrolysis time was 10 h.The prepared enzymatic hydrolysate(< 1 kDa)which prepared in this study could significantly reduce the content of TG in cells.3.In vitro antioxidant results indicated that CSCP had good scavenging activity toward to DPPH,hydroxyl,ABTS and superoxide anion radical,indicating that CSCP had good antioxidant activity.4.The results of in vitro cell experiments showed the contents of AST,ALT,MDA and ROS in the cells of the CSCP treated group were decreased when compared with the model group,and showed a dose-dependent relationship.In addition,the contents of SOD,GSH and CAT increased when compared with the model group.TEM results showed that the normal group had more microvilli,while the number of microvilli decreased in the model group.The contents of lipid drops and the number of microvilli in the cells of the dosing group were significantly decreased,especially in the high-dose group.5.In vivo mouse experiment results showed that CSCP could reduce the contents of AST,ALT,DLD and TG in the serum of mice in the model group,increase the content of HLD,and decrease the content of SOD in liver homogenate.The results of HE staining showed that there were more vacuolar lipid droplets in the hepatocytes of the model group,the positive drug group and the CSCP drug group were significantly improved,and the liver cable structure and hepatocyte morphology were close to normal.TEM results showed that there were many round fat droplets with low electron density in the hepatocytes of the model group,and the lipid droplets in the CSCP group and the positive drug group were significantly reduced,and the mitochondria were increased,which had obvious effects on the repair of liver tissue.Experimental conclusion: the NAFLD model of Chang liver cells was established using the 0.25 mM of oleic acid after 24 h induction.The CSCP(< 1 kDa)was prepared by using alkaline protease and could effectively reduce the contents of AST,ALT,MDA and ROS in the NAFLD cell model,and increase the contents of SOD,GSH and CAT in the NAFLD cell model.The CSCP could remove the harmful free radical and show the good antioxidant activities.Meanwhile,the degree of liver tissue lesions in the mouse NAFLD model induced by high-fat diet was reduced when treated with CSCP,so CSCP that prepared in this study has the better repair effect on NFALD.