Effects Of Xiaoyao Pill On Intestinal Metabolites And Fatty Acid β-Oxidation In Rats With Liver Depression And Spleen Deficiency Syndrome Of Non-Alcoholic Steatohepatitis

ObjectiveBy studying the effect of Xiaoyao pill on the changes of fecal endogenous metabolites and fatty acidβoxidation in nonalcoholic steatohepatitis（NASH）rat with liver depression and spleen deficiency syndrome,the mechanism of Xiaoyao pill was discussed.MethodsTwenty-four male Sprague-Dawley rats were randomly divided into normal group,model group and treatment group,with 8 rats in each group.The rats were given high-fat diet,CCl₄subcutaneous injection on the back,hunger disorder and tail clamping,and combined stimulation for 4 weeks to establish an animal model of NASH with liver depression and spleen deficiency syndrome.After the model was successfully established,the treatment group was given Xiaoyao pill aqueous solution by gavage,and the remaining rats were given the same amount of normal saline,once a day for 4 weeks.During this period,the generally behavioral characteristics of rats in each group were observed.After the administration of the drug,the specimens were taken.The histopathology of the rat liver were observed by hematoxylin-eosin staining（HE）and oil red O staining,and semi-quantitative analysis was performed.The levels of alanine aminotransferase（ALT）,total cholesterol（T-CHO）,triglycerides（TG）and low density lipoprotein cholesterol（LDL-C）in rat serum were detected by microplate method.The contents of noradrenaline（NE）and 5-hydroxytryptamine（5-HT）in hippocampus and the content of Interleukin（IL）-1β,IL-6,IL-10 and TNF-αin serum were detected by Elisa.High-throughput liquid chromatography mass spectrometry（LC-MS）was used to analyze the fecal metabolite profiles of rats in each group,which the metabolic pathways involved in the differential metabolites were analyzed.Western Blot,immunohistochemistry（IHC）and immunofluorescence（IF）were used to quantitatively and locatively detect the expression of peroxisome proliferator-activated receptorα（PPARα）,peroxisome proliferators-activated receptor gamma coactivator-1α（PGC-1α）,carnitine palmityltransferase 1α（CPT1α）and acyl-coa oxidase 1（ACOX1）in rat liver tissue.RT-q PCR was used to detect the relative expression levels of PPARαm RNA,PGC1αm RNA,CPT1αm RNA and ACOX1m RNA in liver tissues.Results1.General condition and syndrome index of liver depression and spleen deficiency of rats in each groupDuring the experiment,the rats in the control group had stable mood,shiny hair,normal food intake and stool.The rats in the model group had dull and dull hair,early irritability,late depression,lack of food and drink,tiredness and laziness,loose stool,etc.,which showed many symptoms similar to the TCM syndrome of liver depression and spleen deficiency.Compared with the control group,the liver index of rats in the model group was significantly increased（P<0.01）;the body mass index,the contents of 5-HT and NE in the hippocampus were decreased（P<0.05,P<0.01）.Compared with model group,liver index was significantly decreased（P<0.01）;the body mass index,the contents of 5-HT and NE of hippocampus were increased（P<0.05,P<0.01）.2.Results of liver histopathology in each groupUnder the light microscope,HE staining and oil red O staining of liver tissue sections of rats showed that the hepatocytes in the control group were orderly arranged,the cell cords were radially distributed,the morphology of hepatic sinusoids was normal,and the nucleus was clearly visible.Compared with the control group,the hepatocytes in the model group were swollen,with a large number of round or round-like vacuoles in the cytoplasm with smooth boundaries,squeezing the nucleus to the edge,shaped like a ring,and a large number of inflammatory cells aggregated.Compared with model group,the liver cells in the treatment group were slightly edematous,the number of fat vacuoles were significantly reduced,and a very small amount of inflammatory cell infiltration was observed.The results of histological observation were further confirmed by semi-quantitative statistical analysis of non-alcoholic fatty liver disease NAS score and oil red O staining positive area.3.Serum biochemical indexes and inflammatory factors were detected in each groupCompared with the control group,the contents of ALT,T-CHO,TG,LDL-C,IL-1β,IL-6 and TNF-αin serum of rats in model group were significantly increased（P<0.01）,while IL-10 was significantly decreased（P<0.01）.Xiaoyao Pill had significant regulating effect on the above indexes in the model group（P<0.05,P<0.01）.4.Fecal metabolomics results of rats in each groupCompared with the control group,57 metabolites in feces of rats in model group had significant changes,and 25 of them,including acetyl-L-carnitine,histamine,pyruvate,L-serine and cytosine,were significantly improved after Xiaoyao Pill intervention（P<0.05,P<0.01）,which mainly involved 5 metabolic pathways:Histidine metabolism,arginine synthesis,purine metabolism,cysteine and methionine metabolism,phenylalanine metabolism.5.Expression of key enzymes in fatty acidβ-oxidation pathway in liver tissue of rats in each groupCompared with the control group,the transcription and expression of PPARα,PGC1α,ACOX1 and CPT1αin liver cells of the model group were decreased（P<0.01）.However,these indexes were increased in Xiaoyao Pill group（P<0.05,P<0.01）.The results of IHC and IF of rat liver tissue also confirmed that Xiaoyao Pill had up-regulation effect on the above indexes in the model group.Conclusion1.Xiaoyao Pill can improve liver lipid accumulation,dyslipidemia and liver function injury in NASH rats with liver depression and spleen deficiency syndrome,and regulate endogenous differential metabolites in feces.2.The therapeutic mechanism of Xiaoyao Pill may be related to the up-regulation of the expressions of PPARα,PGC1α,ACOX1 and CPT1αin liver tissue,and the promotion of liver fatty acidβoxidative metabolism.3.Xiaoyao pill regulates the contents and types of endogenous metabolites in feces of NASH rats with liver depression and spleen deficiency syndrome,which may be achieved by regulating fatty acidβ-oxidation.