Protective Effect Of Natto Combined With Red Yeast Rice On Alcoholic Liver Injury In Rats

ObjectiveIn this study,natto combined with red yeast rice was used as an intervention to observe its protective effect on alcoholic liver disease(ALD)rats,and its protection mechanism was preliminarily discussed.Method1.Modeling and grouping: Forty-eight SPF male Wistar rats were adaptively fed for one week,then randomly divided into 4 groups according to body weight: Control group,with normal saline by gavage;Model group,with a liquor containing 56% alcohol at an dosage of 6ml/kg·bw·d for 1 week + 8ml/kg·bw·d for 1 week + 10ml/kg·bw·d for 1 week + 11 ml/kg·bw·d for 7 weeks by gavage;natto combined with red yeast rice group,with natto combined with red yeast rice(containing 595.59 FU/kg·bw·d and 4.46 mg/kg lovastatin)by gavage 1 hour before the liquor treatment as the mode;diammonium glycyrrhizinate group(positive control group),with diammonium glycyrrhizinate at dosage of 200 mg/kg·bw·d.1 hour before the liquor treatment as the model.The gavage dose of alcohol in the two intervention groups was the same as that in the model group,and the experiment lasted for 10 weeks.Rats were fasted for 12 h after the last gavage,anaesthetized with 3% barbaric sodium,blood was taken from abdominal aorta,and serum was separated for biochemical indicators detection.Frash liver and small intestine tissues were quickly removed,and were fixed for histopathological and ultrastructural observation,the remaining tissues were stored at-80? for testing.2.Hepatoxylin-eosin staining(H&E)was used to observe the pathology of rat liver and small intestine.Transmission electron microscope was used to observe the ultrastructural changes of liver and small intestine of rats.3.Automatic biochemical analyzer was used to detect the activities of glutamic pyruvic transaminase(ALT),glutamic oxaloacetic transaminase(AST),glutamyl transpeptidase(GGT)and alkaline phosphatase(ALP)in serum of rats.4.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum endotoxin expression levels of rat.5.Western Blot(WB)was used to detect the expression levels of Toll-like receptors 4(TLR4),cluster of differentiation-14(CD14)and tumor necrosis factor alpha(TNF-?)in liver tissues of rats.Result1.Natto combined with red yeast rice on liver histopathological changes of alcohol-exposed rats.H&E staining results showed that,in normal control group,the structure of hepatic lobule was complete,the hepatocyte cords were arranged radially around the central vein,and the cytoplasm was full.In model group,the hepatic lobule structure was fuzzy,the liver cell arrangement was disordered,the cytoplasm was shrunken and accompanied by diffuse cytoplasmic vacuoles and steatosis.A large number of inflammatory cell infiltration were observed in portal area.The above pathological changes in natto combined with red yeast rice intervention group and diammonium glycyrrhizinate intervention group were significantly relieved.The results of TEM observation showed that,in normal control group,the hepatocyte nuclei of rats were round or oval,the nuclear membrane was complete and clear,the rough endoplasmic reticulum was orderly arranged,and ribosomes were abundant.In model group,the hepatocyte nucleus were shrinken and deformed,mitochondria were swelled and ruptured,rough endoplasmic reticulum were expanded and ruptured,and ribosomes degranulate.The above pathological changes were significantly improved in natto combined with red yeast rice group and diammonium glycyrrhizinate group,and the improvement effect in natto combined with red yeast rice group was lower than that in diammonium glycyrrhizinate group.2.Natto combined with red yeast rice on small intestine pathological changes of alcohol-exposed rats.H&E staining results showed that,in control group,the small intestinal villi of rats were arranged in order and with complete morphology,uniform submucosal glands and no inflammatory cell infiltration.In model group,small intestinal villi were detached and arranged disorderly,submucosal glands atrophied and inflammatory cell infiltration was observed.The above pathological changes were significantly improved in natto combined with red yeast rice group and diammonium glycyrrhizinate group.The TEM observation of small intestine showed that,in normal control group,the intestinal microvilli were slender and abundant,and the cell connections were tight and clear.In model group,the small intestinal microvilli were short and sparse,some of them showed atrophy and shedding,tight junctions swelling and a decrease in the number of desmosomes.The above pathological changes were improved to different degrees in natto combined with red yeast rice group and diammonium glycyrrhizinate group.3.Effect of natto combined with red yeast rice on liver function of alcohol-exposed rats.The results showed that,compared with normal control group,the serum ALT,AST,GGT and ALP activities in model group were increased significantly(P<0.05).After supplementation with natto combined with red yeast rice and diammonium glycyrrhizinate,the activities of serum ALT,AST,GGT and ALP were significantly inhibited(P<0.05).And there was no significant difference in the above enzyme activities between natto combined with red yeast rice group and diammonium glycyrrhizinate group.4.Effect of natto combined with red yeast rice on serum endotoxin levels of alcohol-exposed rats.The results of ELISA showed that,compared with normal control group,the serum endotoxin levels in model group were increased significantly(P<0.05).After supplementation with natto combined with red yeast rice and diammonium glycyrrhizinate,the levels of endotoxin in serum of rats were decreased significantly(P<0.05).And there was no significant difference between natto combined with red yeast rice group and diammonium glycyrrhizinate group.5.Effect of Natto combined with Red yeast rice on the expression of inflammation-related protein of ALD rats.5.Effect of natto combined with red yeast rice on the expression of inflammation-related protein in liver tissues of alcohol-exposed rats.Western blot analysis showed that,compared with normal control group,the expressions levels of TLR4,CD14 and TNF-? protein of liver tissues in model group were significantly up-regulated(P<0.05).After supplementation with natto combined with red yeast rice and diammonium glycyrrhizinate,TLR4,CD14 and TNF-? protein expressions levels of liver tissues were significantly inhibited(P<0.05).There was no significant difference between natto combined with red yeast rice group and diammonium glycyrrhizinate group.Conclusion1.A certain amount of alcohol intake can cause liver damage in rats.2.Natto combined red yeast rice intervention can protect liver injury induced by alcohol in rats.Its mechanism may be related to the repair of intestinal mucosal barrier,reducing endotoxin intestinal leakage,the up-regulation of the expression levels of inflammatory factors such as TLR4,CD14 and TNF-? in liver tissues by natto combined with red yeast rice.