Perilipin 5 Deficiency Improves The Development Of Nonalcoholic Fatty Liver Disease In Mice Induced By High-Fat And High-fructose Diet

Background and ObjectivesNonalcoholic fatty liver disease(NAFLD)has emerged as a global chronic liver disease,covering a range of pathologic changes from simple steatosis to nonalcoholic steatohepatitis(NASH)and progression to fibrosis and cirrhosis,and even hepatocellular carcinoma.NAFLD is caused by excessive deposition of neutral lipids in liver cells,mainly in the form of triglyceride(TG),which is stored as lipid droplets(LDs).LDs play a central role in the transport of fatty acids(FA).Perilipin 5(Plin 5)on the surface of LDs is highly expressed in oxidized tissues,such as the liver.As the main medium for neutral lipid storage and hydrolysis,Plin 5 controls the lipid balance on the surface of LDs in vivo,and will lead to the occurrence of fatty liver when the imbalance occurs.Studies have shown that overexpression of Plin 5 can promote lipid storage in hepatocytes,so Plin 5 plays an important role in the occurrence and development of NAFLD.Our previous in vitro studies have shown that Plin 5 can promote the formation of LDs,inhibit the activation of hepatic stellate cells(HSCs),and induce the apoptosis of the activated HSCs,which provides new insights for the improvement of fatty liver.However,in vitro and in vivo are two completely different research systems.In this study,Plin 5-/-mice were selected as research subjects to evaluate the effect of Plin 5 on NAFLD induced by high-fat and high-fructose diet in vivo.Methods(1)The knockout mice purchased were the Plin 5 gene knockout using the CRISPR/Cas9 technology.Hybridization of female Plin 5+/-and male Plin 5+/-were selected in the offspring,and genotype identification was carried out by agarose electrophoresis.Real-time PCR and Western Blot again verified whether Plin 5 was successfully knocked out.(2)Male C57BL/6J and Plin5-/-mice were randomly divided into 4 experimental groups.The normal diet group was recorded as ND group,and the high-fat and high-fructose diet group as HFD group,which were denoted as ND-C57BL/6J,ND-Plin5-/-,HFD-C57BL/6J and HFD-Plin5-/-,respectively.Intraperitoneally injected glucose tolerance test(IPGTT)was performed in mice one week before execution.(3)All group mice were sacrificed after feeding for 20 weeks,and the general indicators of the mice and the general view of the liver were compared.(4)HE staining was used to observe the liver steatosis.Sirius red staining and Masson staining were used to observe the liver fibrosis.(5)The relative expression levels of Perilipin family members,liver fatty acid binding protein(L-Fabp)and lipid synthesis related factors in the high-fat high-fructose diet group were analyzed by Real-time PCR.The expression levels of proteins related to fatty liver and fibrosis progression were analyzed by Western Blot.Results(1)The purchased mice were successfully knocked out of the Plin 5 gene and hybrid breeding successfully.Animal models of NAFLD were successfully established after 20 weeks of high fat and high fructose diet.(2)Impaired glucose tolerance was observed in mice on the high-fat and high-fructose diet,but the loss of Plin 5 reduced the extent of impaired glucose tolerance(P<0.05).(3)All group mice vital signs were stable,the levels of body weight,weight gain,liver weight,liver weight/body weight,fat and fat/body weight of HFD-Plin 5-/-group decreased(P<0.05).The liver morphology of HFD-C57BL/6J mice showed significant steatosis,while Plin 5 deficiency reduced the degree of steatosis in the liver.(4)By HE staining,it was found that the hepatic steatosis and inflammatory cells infiltration were seen in the whole field,while the loss of Plin 5 reduced the degree of steatosis,with fewer LDs and smaller diameter(P<0.001).In the portal area and around the central vein of the HFD-C57BL/6J group,Sirius red staining showed a large amount of red collagen proliferation,and Masson staining showed a large number of blue collagen fiber hyperplasia,while the loss of Plin 5 reduced the degree of fibrosis(P<0.05).(5)Compared with HFD-C57BL/6J group,the expression levels of the Perilipin family members Plin 3,Plin 4,Plin 5 mRNA,lipid transport protein L-Fabp and lipid synthetic protein SREBP-1c and HMG CoA in the loss of Plin 5 group down-regulated(P<0.05),and TIMP4 and LGALS3 proteins associated with fatty liver and fibrosis progression in liver down-regulated(P<0.05).ConclusionThe high-fat and high-fructose diet leads to the disorder of glucose and lipid metabolism,liver steatosis and fibrosis in mice,which induces the occurrence and development of NAFLD in mice.The loss of Plin 5 can reduce the degree of steatosis and fibrosis in mice and delay the development of NAFLD by regulating the expression of lipid metabolism-related genes such as lipid synthesis.