| Objective In this study,the classic Gao-Binge model was used to establish a mouse alcohol-induced liver injury model with Lieber-De Carli alcohol or a control liquid feed.Our aim is to investigate the role of NK cells in alcoholic liver steatosis,and then provide some research evidence for pathogenesis and immune-targeted therapy in alcoholic liver steatosis.Methods Six to eight-week-old wild-type C57BL/6J mice were fed with Lieber-De Carli alcohol or control liquid feed.The mice were randomly divided into control group（Control）,alcohol group（Et OH）,and alcohol+NK depletion group.（Et OH+anti-As GM1）,alcohol+NK activation group（Et OH+poly I:C）.Mice in the control group were fed with control liquid feed,and mice in the alcohol group were fed with alcohol liquid feed containing 5%（v/v）alcohol.The mice in the Et OH+As GM1 group were received continously intraperitoneal injection of anti-As AM1 antibody every three days to deplete NK cells at dose of 50ug per mouse every time on the basis of alcohol feeding.The mice in Et OH+poly I:C group were injected intraperitoneally with poly I:C at a dose of 20 mg/kg/time,once every 2 days.The weight of the mice and the consumption of liquid feed were recorded daily.The mice in the control group were fed according to the isocaloric diet consumed by the mice in the alcohol group to ensure that the mice in all groups ingested the same calories.The mice in the alcohol group were given 31.5%alcohol by gavage at a dose of 5 g/kg on the morning of 11thday,and the mice in the control group were given the same amount of maltodextrin as control.Nine hours after gavage,mouse serum was collected for detection of alanine aminotransferase（ALT）and triglyceride（TG）,and liver tissue was harvested for TG mesurement,m RNA detection by quantitative PCR,cell analysis by flow cytometry and liver histology,such as HE staining and oi red O staining.Results After feeding with alcohol,serum level of ALT increased significantly,and HE staining showed that the liver tissue was infiltrated by inflammatory cells,indicating that the liver was in an inflammatory injury state.In addition,both of TG content in serum and fat vacuoles and lipid droplets in liver tissues increased significantly.Compared with the control group,the proportion and absolute number of NK cells in liver mononuclear cells of the mice in alcohol group decreased significantly,suggesting that NK cells may be involved in the development of alcoholic liver disease.Among the mice fed with alcohol,depletion of NK cells by intraperitoneal injection with anti-As GM1 antibody resulted in significant reduction of liver TG levels,liver steatosis,and m RNA levels of genes that encode proinflammatory factors and fat synthesis-related enzymes.These data suggested that the absence of NK cells could,to some extent,restore alcohol-induced liver steatosis.Compared with the control group,not only the proportion and absolute number of NK cells in the alcohol group decreased significantly,but also the composition of their cell subpopulations also changed obviously,showing the ratio of c NK cells decreased significantly but the ratio of Lr NK cells increased significantly.To verify the role of NK cells in alcoholic liver steatosis,mice were injected with poly I:C intraperitoneally to activate NK cells.This treatment leaded to decreased liver TG level and liver lipids shown by oil red O staining,but increased the percentage of c NK cells in NK cell subsets,indicating that the increase of c NK cells after NK cell activation can attenuate alcohol-induced liver steatosis.In order to further verify that c NK cells can inhibit alcoholic hepatic steatosis,adoptive transfer of c NK cells was used to increase the number of c NK cells in the liver of recipient mice.Pathological results showed that liver steatosis was significantly weakened after transfer of c NK cells,indicating that c NK cells does play an important protective role in alcohol-induced hepatic steatosis.In addition,we analyzed the expression of activated and inhibitory receptors on NK cells.The expression of ly49 family receptors on c NK cells in alcohol group was significantly lower than that in the control group,and the expression levels of ly49I and ly49H on Lr NK cells in alcohol group were much lower than those in the control group.In addition,PD-1 and LAG-3 on c NK cells in the alcohol group were significantly higher than those in the control group,while the expression of inhibitory receptors on Lr NK cells was not affected.Conclusion c NK cells play a protective role in alcohol-induced liver steatosis,and increased c NK cells significantly attenuates alcohol-induced liver steatosis.The mechanism of alcohol-induced liver injury may be related to the lack of c NK cells which results in unbalanced activated and inhibitory receptors on NK cells and accumulation of inflammatory cytokines. |
