| Objective:To explore the influence of the compatibility of berberine and evodiamine,the main components of coptidis and evodia,on the blood lipid level and serum cholesterol metabolites markers level in hyperlipidemia rats induced by high-fat diet and to reveal the mechanism of the compatibility of Coptis and evodia on reducing blood lipid by studying the molecular mechanism of inhibiting cholesterol absorption pathway SIRT1/NPC1L1-ACAT2-Apo B48.Methods:Sixty-four male healthy Sprague-Dawley rats were randomly divided into blank group(n =8)and model group(n=56)according to the level of TC and TG.After 4 weeks fed with high-fat diet,they were randomly divided into 7 groups,model group,atorvastatin group(20mg/kg),ezetimibe group(10mg/kg),berberine group(72.8mg/kg),evodiamine group(16.8 mg/kg),low(89.1 mg/kg)and high(178.2mg/kg)dose of the compatibility of coptidis and evodia group.All the animals were fed the corresponding concentration and volume of drugs by intragastric administra-tion every day,dosage volume 10 ml/kg,continuous lavage for 4 weeks.1.Assay for serum lipids: Every week,blood samples were collected from the tail veins of the rats from the beginning of eating high-fat diet to the end of drug intervention.Approximately,0.5 ml of blood was taken from each rat.The tubes were then centrifuged at 3500 r for 15 min.Collected serum samples were analysed to determine serum TC,TG,HDL-C and LDL-C using a commercial kit.2.The liver liver pathology morphological observation: The left lobe of the liver was stained with HE.After pruning,dehydration,embedding,slicing,HE staining,mounting,Then placed under the microscope to observe and the analyzed with Motic Images Advanced.3.Measurements of cholesterol absorption markers: After the last drug lavage,the blood was obtained from the abdominal aorta,then centrifuged.Campesterol,stigmas-terol,sitosterol were measured by GC-MS.4.Using Western Blot method to test the liver SIRT1 protein expression in each group of rat.5.Using Immunohistochemical staining SP method to detect the expression of the rat small intestine NPC1L1,ACAT2 and Apo B48 in each group.Results:1.Coptis and evodia compatibility relieve the blood lipid level in hyperlipidemia model rats Compared with the blank group,the levels of serum TC and LDL-C were significantly increased(P<0.01);but the content of Serum TG and HDL-C has no obvious difference(P>0.05)in the model group rats,indicating the model is successful.Compared with the model group,the level of serum TC,TG and LDL-C were significantly lower(P<0.05),yet serum levels of HDL-C has no obvious difference(P>0.05)in the all drug intervention groups;Compared with the Berberine or evomine group alone,the low dose of coptis and evodia compatibility group has lower serum TC,TG and LDL-C.2.Coptis and evodia compatibility improved the rat liver pathological morphology The hepatocytes of the blank group rats had no steatosis and fibrosis hyperplasia,and the hepatic cord was arranged neatly.The hepatic sinus and the central vein were not congested and the inflammatory cells were formed.In the model group,the hepatocytes of the model group rats had different degrees of steatosis,the hepatic fibrosis hyperplasia was obvious,the sinusoidal expansion was congested,and a small amount of inflammatory cell infiltration was found around the hepatic sinus and the central vein.Compared with the model group,the liver cell steatosis and liver fibrosis in each administration group were reduced to different degrees.The improvement of hepatic steatosis and fibrosis in the coptis and evodia compatibility group was stronger than that of the single group.3.Coptis and evodia compatibility reduced the level of serum cholesterol absorption markers in hyperlipidemia model rats Serum stigmasterol and sitosterol in the model group were remarkably higher(P<0.01)than in blank group,which suggested abnormal cholesterol absorption existing in hyperlipidemia rats.Drug administration prominently alleviated the elevation of serum stigmasterol(P<0.05)and sitosterol(P<0.01),but did not measured serum campesterol.And the level of serum sitosterol in the compatibility group is lower than that of berberine group or evodiamine group alone,but does not reflect the advantages in reducing the stigmasterol level.4.Coptis and evodia compatibility enhanced expression of rat liver SIRT1 Compared with the blank group,model group rats liver SIRT1(P<0.01)protein expression were lower.Compared with the model group,all groups of drugs rats liver SIRT1 protein expression were higher.The level of SIRT1 was significantly higher(P<0.05)in the group of evodiamine and the compatibility of coptis and evodia group.And the level of SIRT1 in the coptis and evodia compatibility group was stronger than the berberine or evodiamine alone.5.Coptis and evodia compatibility prevented the expression of small intestine NPC1L1,ACAT2 and Apo B48 protein in hyperlipidemia rat.Compared with the blank group,the model group rats had higher expression of small intestine NPC1L1,ACAT2,Apo B48(P<0.01).Compared with model group,the level of these 3 protein in all drug groups showed a downward trend.The berberine and the evodiamine treatment alone did decrease NPC1L1(P<0.01)level remarkbaly,but did not reduce the expression of ACAT2 and Apo B48 evidently(P>0.05),high and low dose group of coptis and evodia compatibility lessen the expression of intestine NPC1L1,ACAT2,Apo B48 obviously(P<0.01).And the effect of two dose groups in compatibility were stronger than the berberine or evodiamine alone.Conclusion:1.Coptis and evodia compatibility group can reduce hyperlipidemia rats blood lipid levels,improve the level of cholesterol absorption markers and hepatic pathological changes.2.Coptis and evodia compatibility group can regulate the SIRT1/NPC1L1-ACAT2-Apo B48 signal pathway and improve the cholesterol absorption.The compatibility of Coptis and evodia in the regulation of lipid-lowering and inhibition of cholesterol absorption is stronger than that of single use,which reflects the scientific connotation of Coptis and evodia compatibility. |
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