| Background:Hyperlipidemia is an abnormality of lipid metabolism,characterized by an elevation of total cholesterol(TC),triglyceride(TG),and low density lipoprotein-cholesterol(LDL-C),and/or a decreasing of high density lipoproteincholesterol(HDL-C)in circulating levels.Hyperlipidemia is also the primary risk factor for the occurrence and development of chronic diseases such as atherosclerosis,heart disease,stroke and fatty liver.More and more studies have found that the body's physiological metabolism and growth and development in addition to the controlled by their genes,the symbiosis in the human body of a large number of microbial genetic information also play an important role,intestinal flora of the body's immune,nutrition and metabolism plays an important role,and closely related with lipid metabolism.Chinese medicine believes that temper clogging,phlegm and damp is the main pathogenesis of hyperlipidemia,three jiao,shengqingjiangzhuo is the key to the treatment of hyperlipidemia.Compatibility of Coptidis Rhizoma and Evodia rutaecarpa is the representative of the theory that embodies the theory of symplectic,bitter and descending compatibility.It has a long history of clinical application.These two herbs can be processed not only by each other but also can be used separately.A large number of clinical and experimental studies have confirmed that the Chinese herbal medicine,Coptidis Rhizoma,Evodia rutaecarpa and its effective components,have a clear effect on reducing fat.In addition,the absorption of small intestine is the basis of the effect of traditional Chinese medicine,and the intestinal flora provides a new target for the study of the mechanism of the Coptidis Rhizoma and Evodia rutaecarpa combinations.The previous study found that the compatibility of Coptidis Rhizoma and Evodia rutaecarpa has a significant effect on the level of blood lipid in hyperlipidemia rats,and the material base of its function is alkaloid.It is demonstrated that the compatibility of Coptidis Rhizoma and Evodia rutaecarpa with the proportions of 1:1has a significant downregulation effect on serum and liver cholesterol levels in highfat model animals.The intestinal microflora is closely related to cholesterol bile acid metabolism.Whether Coptidis Rhizoma and Evodia rutaecarpa combinations with the regulation of intestinal flora expression in hyperlipidemic animals are consistent with the regulation of cholesterol metabolism needs further study.Objective:The hyperlipidemia model rats as the research carrier,to observe the effect of berberine and evodiamine combinations which are the main components of Coptidis Rhizoma and Evodia rutaecarpa combinations,based on cholesterol metabolism in rats with hyperlipidemia related genes and intestinal microflora,and investigate the molecular mechanism of Coptidis Rhizoma and Evodia rutaecarpa compatibility on cholesterol metabolism and its relationship with intestinal flora,reveal the further mechanism of Coptidis Rhizoma and Evodia rutaecarpa compatibility.Methods:1.Molding:Seventy male healthy Sprague-Dawley rats were randomly divided into blank group(n =10)and model group(n=60)according to the level of TC and TG.After 4weeks fed with high-fat diet,48 rat were selected out,randomly divided into 6 groups,model group(n =8),ezetimibe group(n =8),berberine group(n =8),evodiamine group(n =8),low dose of the compatibility of coptidis and evodia group(n =8)and high dose of the compatibility of coptidis and evodia group(n =8).All the animals were fed the corresponding concentration and volume of drugs by intragastric administration everyday,dosage volume 10ml/kg,continuous lavage for 4 weeks.2.Intragastric administration:After 4 weeks of feeding,the 50 HFD induced rats were subdivided into five groups: a group of 10(HFD)was treated with sodium carboxymethyl cellulose(CMC;0.5%);the other four groups were treated with ezetimibe(EZ)(10 mg/kg b.w.),berberine(BB)(72.6 mg/kg b.w.),evodiamine(EV)(16.6 mg/kg b.w.),and combination of berberine and evodiamine(BB+EV)(89.2 mg/kg b.w.).The berberine,evodiamine,and their combination were dissolved in 0.5% CMC.These compounds were administrated to rats by gavage at a dose of 10 ml/kg body weight for fourweeks fed with HFD.3.Determination:(1)Lipid profile analysis: The levels of TC,TG,LDL-C,and HDL-C in rat serum were measured by biochemical method.(2)The liver pathology morphological observation: The left lobe of the liver was stained with HE.(3)Hepatic lipid analysis: The levels of hepatic TC and TG levels were measured by biochemical method.(4)Serum bile acid analysis: The level of bile acid in serum was measured by Enzyme-linked immunosorbent assay.(5)Determination of hepatic genes related with cholesterol metabolism: The mRNAs in hepatic were extracted by reverse transcription-polymerase chain reaction,and the levels of SREBP2,HMGCR,PPAR?,LXR? and CYP7A1 genes were determined.(6)Determination of intestinal genes related with cholesterol metabolism: The mRNAs in intestine were extracted by reverse transcription-polymerase chain reaction,and the levels of LDL-R,NPC1L1,ABCG5,ABCG8,and SR-BI genes were determined.(7)Detection of intestinal microbiota expression: extract the total DNA from feces of each group and carry out quality detection and screening.Then,the Illumina two generation sequencing technology platform was used to make 16 sDNA sequencing of the prepared DNA samples,and the data were sorted,optimized and analyzed.(8)Correlation between intestinal microbiota and physicochemical data analysis:according to the ecology methods on the species abundance of logarithmic transformation and the physicochemical factors of standardization,by using the calculation method of Spearman in different groups on the degree of correlation calculation of each species with each physicochemical index and significant test.Results:1.Effect of BB and EV compatibility on lipid profiles in hyperlipidemia ratsBy comparison with CON group,meaningful elevation of serum TC and LDL-C was observed in the HFD-fed rats in week 4(P<0.05 or P<0.01).The serum levels ofTC,TG,and LDL-C were significantly decreased in group BB,EV,and BB+EV compared with those in HFD group in week 8(P<0.05 or P<0.01).2.Effect of BB and EV compatibility on the liver pathomorphology of hyperlipidemia ratsThe rats in CON group showed a well-structured liver.HFD rats manifested pathological changes of the liver manifested swollen hepatocytes coupled with hepatocytes steatosis in the form of increasing degree of vacuolar degeneration.By comparison of HFD group,these changes could be obviously alleviated by EZ,BB,and BB+EV.3.Effect of BB and EV compatibility on hepatic lipids in hyperlipidemia ratsHepatic TC content in BBEVL and BBEVH group were remarkable decreased compared with the HFD group(P<0.05).4.Effect of BB and EV compatibility on serum bile acid in hyperlipidemia ratsBy comparison with CON group,meaningful elevation of serum bile acid was observed in the HFD-fed rats(P<0.05).The serum level of serum bile acid was significantly decreased in EV group and BBEVH group compared with those in HFD group(P<0.05).5.Effect of BB and EV compatibility on hepatic genes related with cholesterol metabolism in hyperlipidemia ratsThe level of hepatic gene levels of SREBP2 and HMGCR were significantly decreased in BB group and BBEVL group compared with those in HFD group,while The level of hepatic gene levels of PPAR?,LXR? and CYP7A1 in BB group and BBEVL group were accelerated significantly compared with those in HFD group.6.Effect of BB and EV compatibility on intestinal genes related with cholesterol metabolism in hyperlipidemia ratsIt was demonstrated that BB,BBEVL and BBEVH decreased the intestinal NPC1L1 gene level in hyperlipidemia rats,while increasing intestinal SR-BI gene level.By comparison with HFD group,decease of intestinal ABCG5 and ABCG8 gene levels were observed in BB group and BBEVL group.Treatment with BB and BBEVL tended to increase the expression of intestinal LDL-R gene,although just in the edge of significance(P>0.05).7.Effect of BB and EV compatibility on intestinal microbiota expression in hyperlipidemia ratsCompared with normal diet group,the diversity of gut microflora in the high fat diet group decreased significantly,which can be improved by BB and EV combination.The effects of BB and EV compatibility on the diversity of gut microflora included increasing the expression of Lactobacillus and other beneficial microorganisms,reducing the expressions of harmful bacteria such as Desulfovibrio,Blautia,Prevotella,Lachnoclostridium and so on,and increasing the expression of intestinal barrier repair bacteria,such as Akkermansia.As the improvement of bacteria in the intestinal barrier repairment,the low dose of BB and EV compatibility is better than the single use of berberine or evodiamine.8.Correlation analysis of intestinal microflora and cholesterol metabolism related indexes in hyperlipidemia rats of each groupAt the level of the genus,the positive associations with serum cholesterol and serum LDL cholesterol levels are Ruminococcus_gauvreauii_group,Allobaculum,Lachnoclostridium,Blautia,Eubacterium_hallii_group,Intestinibacter,Peptoclostridium,Escherichia_Shigella,and Nitrosomonas.The positive associations with serum bile acid levels are Ruminococcus_gauvreauii_group,Allobaculum,Parasutterella,Escherichia_Shigella,Nitrosomona,and Plesiomonas.Conclusion:1.The compatibility of Coptidis Rhizoma and Evodia rutaecarpa have a clear effect on lipid lowering in experimental hyperlipidemic rats,which is mainly reflected in lowering serum lipids,improving liver function and regulating the expression level of bile acids.2.The effect of compatibility of Coptidis Rhizoma and Evodia rutaecarpa on the cholesterol metabolism related genes in high fat model is mainly reflected in three aspects: inhibiting liver cholesterol synthesis,promoting cholesterol decomposition,and inhibiting the absorption of cholesterol in small intestine.3.The compatibility of Coptidis Rhizoma and Evodia rutaecarpa can increase the diversity of intestinal flora in the high fat model rats,reduce the condition pathogenicbacteria and increase the intestinal barrier repair bacteria.Compared with the two groups,the compatibility group was more able to restore the diversity of the intestinal flora in the high fat model rats. |
PDF Full Text Request