| ObjectiveBy establishing a mouse model of alcoholic liver disease,the effects of Jianpi Huoxue Recipe on liver function,liver pathology,plasma endotoxin,plasma TNF-α and intestinal barrier of mice with alcoholic liver disease were studied,and the specific targets and mechanism of Jianpi Huoxue Recipe in the treatment of alcoholic liver disease were explained..Methods1.Experiment 1:Pre experiment to establish the mouse model of alcoholic liver disease and its evaluationTwenty C57BL/6N male mice were randomly divided into normal group and model group.Each group of 10 mice were given Lieber-decalli control liquid diet on the first-fifth day after one week adaptive feeding,Lieber-decalli control liquid diet was given to the normal mice on the 6th-15th day,Lieber-decalli control liquid diet was given to the mice in the model group,and the model group mice were given Lieber-decalli alcohol diet,and the model group mice were given high-dose alcohol diet in the morning of the 16th day The rats in the normal group were given equal heat maltose solution(9g/kg)and the model was finished after 9 hours.The plasma and liver tissues of mice were collected.The success of the model was evaluated by observing the overall state of mice,recording the changes of body weight,detecting AST,ALT level of plasma liver function index and he staining of liver.2.Experiment 2:The effect of Jianpi Huoxue Recipe on liver index,liver function index and liver pathology in alcoholic liver disease model miceForty C57b1/6n male mice were randomly divided into normal group,model group,glutamine group,spleen invigorating and Activating Blood Recipe group.Each group of 10 mice was given a week of adaptive feeding.The mice model of alcoholic liver disease was established by using Lieber-decalli alcohol liquid diet.Each group was given the same volume of normal and model groups,and glutamine group was given glutamine solution(220mg/kg)was given stomach,and the spleen invigorating and Activating Blood Decoction(16g/kg)was given to the group of Jianpi Huoxue Decoction(16g/kg),once a day,and was given for 8 days.After the model was completed,the plasma and liver tissues of mice were collected.The liver index of each group of mice was calculated,AST and ALT were detected by automatic biochemical analyzer.HE staining was used to observe the pathological changes of liver,and the therapeutic effect of Jianpi Huoxue recipe was verified.3.Experiment 3:The intervention effect and mechanism of Jianpi Huoxue Recipe on alcoholic liver disease model miceThe same as experiment 2,the plasma and small intestine tissues of each group were collected.The levels of TNF-α in the plasma were detected by ELISA,the endotoxin level was detected by spectrophotometry,the pathological changes of the small intestine were observed by HE staining,and western western blotting was used Blot)to detect the expression of close junction ZO-1 and occludin in small intestine tissues,reverse transcriptase PCR to detect the changes of the genes of close junction ZO-1 and occludin in small intestine,and to explore the specific mechanism and target of Jianpi Huoxue Recipe in the treatment of alcoholic liver disease.Results1.Experiment 1:Establishment and evaluation of mouse model of alcoholic liver diseaseThere was no significant difference in the body weight between the model group and the normal group before and after modeling(P>0.05),which confirmed that the normal group and the model group were fed with equal calories during the modeling period;compared with the normal group,the overall state of the model group was poor,the liver index was significantly increased(P<0.05),the liver function indexes AST and ALT were significantly increased(P<0.01),and the liver pathology of the normal group was improved Objective:to establish a mouse model of alcoholic liver disease(ALD).Methods:the pathological changes of liver in model group were as follows:the nucleus was round and clear,the hepatic cords were arranged in order,and there was no obvious change.The pathological changes of liver in model group included a large number of steatosis,disordered arrangement of hepatocytes,unclear boundary of hepatocytes,unclear structure of hepatic lobules,and inflammatory cell infiltration2.Experiment 2:Jianpi Huoxue recipe can improve liver index,liver function index and liver pathology of mice with alcoholic liver diseaseCompared with the normal group,the liver index of the model group increased significantly(P<0.01),and the liver index of the glutamine group and Jianpi Huoxue Formula group decreased significantly(P<0.05);compared with the normal group,the liver index of the model group decreased significantly(P<0.05)Compared with the normal group,the activities of AST and ALT in the plasma of the model group were significantly increased(P<0.01),and compared with the model group,the activities of AST and ALT in the plasma of the glutamine group and the Jianpi Huoxue Formula group were significantly decreased(P<0.05)The histopathological observation of the liver showed that the activities of AST and ALT in the model group were significantly increased(P<0.01)There were a lot of steatosis and inflammatory cell infiltration in the liver.The pathological steatosis and inflammatory cell infiltration in the liver were significantly improved in the glutamine group and the Jianpi Huoxue Formula group.There was no significant difference in the indexes between the Jianpi Huoxue Formula group and the glutamine group.3.Experiment 3:Intervention effect and mechanism of Jianpi Huoxue Recipe on alcoholic liver disease model miceCompared with the normal group,the levels of TNF-α and endotoxin in the model group were significantly increased(P<0.01);the levels of TNF-α and endotoxin in the glutamine group and Jianpi Huoxue Formula group were significantly decreased(P<0.01);compared with the normal group,the intestinal tight junction ZO-1 and occludin protein in the model group were significantly decreased(P<0.01);compared with the model group,the levels of TNF-α and endotoxin in the glutamine group and Jianpi Huoxue Formula group were significantly decreased(P<0.01)The protein levels of ZO-1 and occludin in intestinal tight junction of mice in Jianpi Huoxue Formula group were significantly increased(P<0.05);compared with normal group,the gene levels of ZO-1 and occludin in intestinal tight junction of mice in model group were significantly decreased(P<0.01);compared with model group,the gene levels of ZO-1 and occludin in intestinal tight junction of mice in glutamine group and Jianpihuoxue formula group were significantly increased(P<0.01);the gene levels of ZO-1 and occludin in intestinal tight junction of mice in model group were significantly increased(P<0.01);the protein levels of ZO-1 and occludin in intestinal tight junction of mice Histopathological observation of small intestine showed that in the model group,the morphology of intestinal tissue was disordered,some intestinal glands and villi were damaged,goblet cells and epithelial cells were significantly reduced,and the integrity of epithelial layer was damaged;the intestinal pathology of Jianpi Huoxue Formula group and glutamine group was significantly improved,and there was no statistical difference between Jianpi Huoxue Formula group and glutamine group.Conclusion1.C57BL/6N mice fed with Lieber-DeCarli alcohol liquid diet can successfully establish the mouse model of alcoholic liver disease,which can provide a stable and repeatable animal model for later experiments.2.Jianpi Huoxue Decoction can reduce AST and ALT,improve liver pathology of mice with alcoholic liver disease,and has good therapeutic effect on mice with alcoholic liver disease,and the therapeutic effect is equivalent to that of positive control drug.3.Jianpi Huoxue recipe may reduce the damage of intestinal barrier function,endotoxin and TNF-α level through the mechanism of intestine liver axis,and then reduce liver injury to treat alcoholic liver disease mice. |
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