Expression Of ZAG In NAFLD Mice And Its Effect On Liver Inflammation

Objective: To establish a nonalcoholic fatty liver disease model induced by high fatdiet and observe the dynamic changes of Zinc alpha2glycoprotein.Methods: All of48male C57BL/6mice were randomly divided into high fat diet(HFD) group and standard diet (SD) group. The serum IL-6, IL-8, TNF-α, IL-1β andZAG were assayed by ELISA kit at the4th,8th,12th and16th week. Hepatictriglyceride content was detected through liver homogenate. The histologicalexamination of liver tissues in mice was analyzed by H&E staining. The mRNAexpression level of ZAG in liver was detected by real-time qPCR and its proteinexpression level was separately detected by Western-blotting andImmunohistochemistry.Results: Compared with SD group, the level of TNF-α were significantly increasedin HFD group from the4th week (P<0.05). The weight gain, liver index, hepatictriglyceride content, IL-6, IL-8and IL-1β were increased gradually after the8th week(P<0.05). The mRNA and protein levels of ZAG were gradually decreased from the4th week (P<0.05). From the8th week, the serum ZAG concentration in HFD groupwas decreased compared to SD group (P<0.05).Conclusion: The mouse model of NAFLD could be successfully established byhigh fat diet and the development of NAFLD could be associated with the lowexpression level of ZAG in liver. Objective: To establish a nonalcoholic steatohepatitis model induced by MCD dietand induce overexpression of hepatic ZAG of mice in NASH by means of tail-veininjection of rAAV2-ZAG-CMV-EGFP and investigate the roles of ZAG in hepaticlipid metabolism and inflammation of mice in NAFLD.Methods: All of54male C57BL/6mice aged8weeks were randomly divided intoControl (n=12), MCD+PBS (n=14), MCD+ZAG (n=14) and MCD+GFP group (n=14)after adaptive feeding for one week. Mice in Control group were fed standard diet for8weeks while ones in the rest three groups were fed MCD diet for4weeks. At the4thweek, overexpression of hepatic ZAG was achieved by means of tail-vein injection ofrAAV2-ZAG-CMV-EGFP (2×1011vg per mouse) in MCD+ZAG group mice. Theother groups which fed MCD diet were respcetively injected PBS orrAAV2-CMV-EGFP in same volume. At the8th week, blood and liver tissues of micewere collected after sacrificed. The liver tissues of mice were stained by H&E and OilRed O for histological analysis. The serum FPG, TG, TC, LDL, HDL, TNF-α, IL-6,IL-8, IL-1β and hepatic triglyceride content were assayed by biochemistry analyzer orELISA kit. The mRNA and protein expression levels of ZAG, lipid metabolism andproinflammatory genes in livers were detected by RT-qPCR, Western-blotting andImmunohistochemistry.Results: At the8th week, the level of food intake, weight, liver mass, serum TG, TC,LDL, HDL, FPG were significantly decreased in mice of groups fed MCDdiet(P<0.05). Liver index, ALT, AST, serum TNF-α, IL-6, IL-8, IL-1β and hepatictriglyceride content were significantly increased (P<0.05). Moreover, The mRNA andprotein level of hepatic HSL, PPARα and CPT1A were decreased (P<0.05). In addition, The mRNA and protein level of hepatic TNF-α、IL-1β、ICAM-1、MCP-1、CD68、F4/80were increased (P<0.05)and increased protein level of IL-6、IL-8inlivers(P<0.05). After overexpression of ZAG in mice of NASH, besides serum lowerTG and TC, the liver index, ALT, AST and serum TNF-α、IL-6、IL-1β were alsoreduced(P<0.05), however, serum IL-8concentration were not decreasedsignificantly(P>0.05). Meanwhile the mRNA and protein level of HSL, PPARα andCPT1A in mice of ZAG overexpression were increased(P<0.05) and its TNF-α、IL-1β、ICAM-1、MCP-1、CD68、F4/80were decreased(P<0.05) as well as reducedprotein level of IL-6and IL-8(P<0.05), and histological analysis indicates the degreeof steatosis and inflammation in the livers of ZAG overexpression in mice of NASHwere alleviated significantly.Conclusion: ZAG could alleviate hepatic steatosis and inflammation in murineMCD-induced NAFLD and which could be associated with the role of ZAG inregulation of genes in lipid metabolism and suppression of proinflammatory genes.