| ObjectiveTo establish a rat model of alcoholic liver injury,observe the ameliorative effect of nattokinase on alcoholic liver injury in rats,and preliminarily explore its ameliorative mechanism.Method1.Animal grouping and model establishment:Sixty male Wistar rats were randomly divided into five groups according to body weight after adaptive feeding for a week:Control group,intragastrically administered with normal saline;Nattokinase control group,orally administered with 530FU/(kg·bw·d)Nattokinase;Model group,gavaged with Erguotou liquor(56%alcohol)at 6ml/(kg·bw·d)for the first week,at 8ml/(kg·bw·d)for the second week,at 10ml/(kg·bw·d)for the third week,and at 11ml/(kg·bw·d)for the fourth to the tenth week;Nattokinase intervention group,administered with530FU/(kg·bw·d)Nattokinase and alcohol;Diammonium glycyrrhizinate intervention group,administered with 200mg/(kg·bw·d)diammonium glycyrrhizinate and alcohol.The same dose of alcohol was given to the latter two groups as that for the model group.the experiment lasted for 10 weeks.Fasting for 12 hours after last gavage,pentobarbital sodium was injected intraperitoneally for anesthesia.Blood from abdominal aorta,HE specimens and electron microscopic specimens of the liver were collected,the remaining liver tissue was frozen by liquid nitrogen and stored in a refrigerator at-80?for later use.2.Morphological examination of liver tissue was performed by hematoxylin and eosin staining,and the liver histopathology was scored.The ultrastructure of liver cells was observed by transmission electron microscopy.3.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),glutamyl transpeptidase(GGT),and cholinesterase(CHE)activities were determined.Endotoxin expression in serum of rats was determined by enzyme-linked immunosorbent assay.4.The number of CD4~+and CD8~+T lymphocyte subsets and natural killer(NK)cells in the peripheral blood of rats was determined by flow cytometry.The ratio of CD4~+T cell percentage to CD8~+T cell percentage was calculated.5.The CD14,TLR4 and TNF-?protein levels in liver tissues was determined by Western blot.Result1.Light microscopic observation results,There were no pathological changes in liver tissue of rats in control group and Nattokinase control group.The structure of hepatic lobules in model group was blurred,disturbance of hepatic chordae,cytoplasmic vacuoles and steatosis,a large number of inflammatory cell infiltration,its histopathological score was significantly higher than that of the normal control group(P<0.05).The above pathological changes were significantly improved after intervention with Nattokinase and diammonium glycyrrhizinate,pathological score decreased significantly(P<0.05),and the decrease of Nattokinase intervention group was lower than that of diammonium glycyrrhizinate intervention group.2.Observation results by transmission electron microscopy:There were no pathological changes in liver tissue of rats in control group and Nattokinase control group.Hepatocyte nucleus atrophy and deformation,decrease in mitochondria,the rough endoplasmic reticulum expands and breaks,ribosome exfoliation is sparse in model group.The above pathological changes were significantly improved after Nattokinase intervention,and the improvement effect was lower than that of diammonium glycyrrhizinate intervention group.3.The results of serum biochemical indicators showed that:Compared with the control group,there was no significant change in nattokinase control group,the levels of ALT,AST,GGT in serum of rats in model group increased significantly(P<0.05),while CHE decreased significantly(P<0.05).Compared with model group,the levels of ALT,AST and GGT were decreased significantly(P<0.05),the levels of CHE has a rising trend in Nattokinase intervention group and diammonium glycyrrhizinate intervention group.And there was no significant difference between nattokinase intervention group and diammonium glycyrrhizinate intervention group.4.The results of serum endotoxin indicators showed that:Compared with the control group,there was no significant change in nattokinase control group,the level of serum endotoxin in model group increased significantly(P<0.05).Compared with model group,the level of serum endotoxin decreased significantly in Nattokinase intervention group and diammonium glycyrrhizinate intervention group(P<0.05).And there was no significant difference between nattokinase intervention group and diammonium glycyrrhizinate intervention group.5.Flow cytometry index results:Compared with the control group,there was no significant change in nattokinase control group.The percentage of CD3~+CD4~+T cells,D3~+CD8~+T cells,TCR??~+CD161~+NK cells decreased significantly(P<0.05),the ratio of CD3~+CD4~+T cell percentage to CD3~+CD8~+T cell percentage increased significantly in model group(P<0.05).Compared with model group,the percentage of CD3~+CD4~+T cells and CD3~+CD8~+T cells were increased significantly(P<0.05),the percentage of TCR??~+CD161a~+NK cell was increased in different degrees,the ratio of CD3~+CD4~+T cell percentage to CD3~+CD8~+T cell percentage decreased significantly in Nattokinase intervention group and diammonium glycyrrhizinate intervention group(P<0.05).And there was no significant difference between nattokinase intervention group and diammonium glycyrrhizinate intervention group.6.The results of western blot showed that,Compared with the control group,there was no significant change in nattokinase control group,the expressions of CD14,TLR4 and TNF-?in liver tissues were significantly increased in the model group(P<0.05).Compared with model group,the expression of TLR4 in liver tissue of rats decreased significantly(P<0.05),the expression of CD14 and TNF-?showed a downward trend in Nattokinase intervention group.The expression of TLR4 and TNF-?in liver tissue of rats in diammonium glycyrrhizinate intervention group decreased significantly(P<0.05),and the expression of CD14 decreased to a certain extent.There was no significant difference between nattokinase intervention group and diammonium glycyrrhizinate intervention group.Conclusion1.A certain amount of alcohol intake can cause liver injury.2.Appropriate supplement of nattokinase can improve alcoholic liver injury to some extent.The mechanism may be related to nattokinase regulating the proportion of CD4~+T cells,CD8~+T cells and NK cells in peripheral blood and inhibiting alcohol-induced up-regulation of serum endotoxin and expression of TLR4,CD14 and TNF-?in liver tissue of rats. |
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