| Objective: In this study,we established a rat model of phlegm and dampness induced by different ratios of high-fat diets,interpreted the principles of Chinese medicine from modern scientific perspectives such as general condition,biochemical indexes,pathological sections,intestinal flora and metabolomics,investigated the biological changes of phlegm and dampness induced by different ratios of high-fat diets,and comprehensively evaluated the effects of coix seed on phlegm and dampness rats to provide theoretical basis for the clinical use and product development of coix seed.Methods:1.Establishment of a rat model of phlegm-dampness induced by different ratios of high-fat diets and comprehensive evaluation of the effect of coix seed on itTo establish the animal model of phlegm induced by different ratios of high-fat diets,96 SD rats were randomly grouped: normal group(recorded as group N),high-fat groups 1,2 and 3(recorded as groups F1,F2 and F3),24 rats in each group,male and female,were given control diet(17% fat content),high-fat diet formula 1(27% fat content),high-fat diet formula 2(40% fat content),and high-fat diet formula 3(60% fat content),respectively.High-fat diet formula 3(60% fat content).The normal group was fed with the control diet,and the high-fat groups 1,2and 3 were subdivided into the model group,the raw coix seed group and the French coix seed group,and were fed with the high-fat diet,the diet containing raw coix seed and the diet containing French coix seed,respectively,for 3 months.The effects of different ratios of high-fat diets on rats and coix seeds on general signs,body mass,body length,Lee’s index,body composition,blood pressure,serum biochemical indexes,pathological sections,etc.were observed,and the effects of different ratios of high-fat diets on model rats and coix seeds on high-fat model rats were preliminarily evaluated.2.Effects of different ratios of high-fat diets on intestinal flora of rats and the intervention effect of coix seed16S rRNA sequencing was performed on rat feces to observe the change of rat intestinal flora diversity,further analyze the change of relative abundance of differential flora at the phylum and species taxonomic levels,and use the LEf Se multi-level species difference discrimination analysis function to deeply explore the interaction of differential bacteria in each group.3.Metabolomics-based analysis of the effects of different ratios of high-fat diets on rats and the intervention effect of coix seedThe metabolic analysis of rat plasma samples with different ratios of high-fat model group was performed by UPLC-Q-TOF/MS technique,and the data were screened by principal component analysis,and the substances with Variable Importance for the Projection(VIP)greater than 1 and P-value less than 0.05 were considered as differential metabolites.The metabolites with identified structures were analyzed by Metabo Analyst 5.0 for metabolic pathways,and the effects of different ratios of high-fat diets on rats and the mechanism of action of Coix seed in dispelling dampness were explored from the perspective of substance metabolism.Results:1.Establishment of rat model of phlegm and dampness induced by different ratios of high-fat diets and comprehensive evaluation of the effect of Coix seed on it(1)General signs observation: During the experiment,it was found that compared with the normal group,the high-fat group showed obvious signs of dampness such as curly and yellowish greasy dorsal hair,depression,drowsiness and laziness,holding and laziness,and loose stools.After the administration of Coix seeds,compared with the normal group and the high-fat model group,the rats in each administration group showed different degrees of remission of dampness,which initially indicated that Coix seeds had a certain degree of dampness dispelling effect.(2)Body mass,body length and Lee’s index of rats: The results showed that the growth rate of body mass was directly proportional to the fat content of the feed,and the higher the fat content of the feed,the faster the growth of body mass.The body mass of rats in each high-fat group increased significantly(P < 0.05)compared with the normal group during the modeling period,and the body mass of rats in each group decreased to some extent after the administration of the drug,although there was no significant difference compared with the model group.The body length of rats in the high-fat 3 group increased significantly(P < 0.05)compared with the normal group.After 3 months of Coix seed intervention,the body length of rats in the high-fat 2group decreased significantly,and there was no significant change in the rest of the groups.During the modeling period,the Lee’s index in the high-fat 3 group was significantly higher compared with the normal group(P < 0.05),and the Lee’s index in the male high-fat 1 group decreased significantly compared with the normal group after 1 and 2 months of C.cochinchinensis administration(P < 0.05).(3)Body composition analysis: Body composition testing using bioelectrical impedance technique revealed that the amount of fat,fat removal and body mass index,overall water content,intracellular fluid and extracellular fluid content were significantly higher in the male high-fat 3 group compared with the normal group(P < 0.05),and the extracellular fluid content was significantly higher in the female high-fat 1,2 and 3 groups(P < 0.05).(4)Blood pressure: There was no significant difference in systolic and diastolic blood pressure in each group compared with the normal group(P > 0.05).(5)Serum biochemical indexes: during the modeling period,there were significant differences in HDL cholesterol in female high-fat 1 group,triglycerides,HDL cholesterol and LDL cholesterol in female high-fat 2 and high-fat 3 groups,total cholesterol and LDL cholesterol in each male high-fat group,and triglycerides in male high-fat 3 group compared with the normal group(P < 0.05).After administration,compared with the high-fat 3 model group,both raw Coix seed and French Coix seed could reduce the triglyceride level in high-fat 3group rats(P < 0.05),and the effect of raw Coix seed in reducing triglyceride was more obvious.(6)Blood rheology and blood cell test: the results showed that the whole blood reducing viscosity was significantly higher in the low,medium and high cut levels in the female high-fat model group compared with the normal group(P < 0.05),and the whole blood reducing viscosity was significantly lower in the low,medium and high cut levels in the female high-fat 1administration group compared with the model group(P < 0.05).The blood cell results showed that the platelet content was significantly lower in the female hyperlipid 1 group compared with the normal group(P < 0.01),and there was no significant difference between the rest of the administered groups compared with the model group(P > 0.05).(7)The results of hematoxylin-eosin staining and oil red staining of liver tissues: the hepatocytes in the high-fat model group increased in size and were irregularly arranged,and certain red lipid droplets and lipid infiltration appeared in the cytoplasm of hepatocytes,and round fat vacuoles of different sizes and a small amount of inflammatory cell infiltration appeared,especially in the high-fat 3 group,which was more obvious.The morphology and structure of the rat liver lobules were more or less normal after administration,and the red lipid droplets were significantly reduced,with a very small amount of lipid droplets distributed in some hepatocytes,especially the effect of raw Coix seed was better.2.Effects of different ratios of high-fat diets on intestinal flora of rats and the intervention effect of Coix seed(1)Alpha diversity analysis showed that during the modeling period,the ace index in the high-fat 3 group and the Shannon index in the high-fat 2 group were significantly lower compared with the normal group(P < 0.05).During the administration phase,the chao1 and ace indices of the high-fat 3 model group were significantly lower compared with the normal group(P<0.05).The chao1,ace and Shannon indices were significantly lower in the raw Coix seed group compared with the high-fat 3 model group(P < 0.05),and the ace index was significantly increased in the French Coix seed group(P < 0.05).(2)At the phylum level,the dominant phylum in each high-fat group was the thick-walled phylum and the anthropoid phylum,and the relative abundance of the thick-walled phylum was positively correlated with the feed lipid content at the modeling stage,while the relative abundance of the anthropoid phylum was negatively correlated with the feed lipid content,and the abundance of the thick-walled phylum decreased and the abundance of the anthropoid phylum increased after the administration of the drug.(3)At the species level,the relative abundance of Colidextribacter,Desulfovibrionaceae and Lachnospiraceae＿NK4A136 were positively correlated with feed lipid content,and the relative abundance of Clostridium＿sensu＿stricto＿1,Muribaculaceae and Dubosiella were negatively correlated with feed lipid content.Compared with the normal group,the relative abundance of Colidextribacter,Desulfovibrionaceae and Lachnospiraceae＿NK4A136 was significantly(P < 0.05)increased in the high-fat 3 group,and the relative abundance of Clostridium＿sensu＿stricto＿1,Muribaculaceae and Dubosiella showed a significant decrease in relative abundance(P < 0.05).During the administration phase,the abundance of Romboutsia and Lachnospiraceae colonies was significantly higher(P < 0.05)in the high-fat3 model group and significantly lower(P < 0.05)in the Romboutsia and Lachnospiraceae abundance in the raw Coix seed group compared with the normal group.(4)Correlation analysis showed that the relative abundance of Colidextribacter,Desulfovibrionaceae and Lachnospiraceae＿NK4A136 was negatively correlated with HDL cholesterol at the modeling stage,and with body weight,Lee’s index,body composition index(overall water content,intracellular water content,extracellular water content,adiposity,non-adiposity,body mass index),total cholesterol,triglycerides,LDL cholesterol,fasting glucose,and glycated serum protein of glycolipid-related indicators were positively correlated.The relative abundance of Muribaculaceae,Clostridium＿sensu＿stricto＿1 and Dubosiella,on the other hand,correlated exactly with body weight,Lee’s index,the above-mentioned body composition indices,total cholesterol,triglycerides,LDL cholesterol,fasting glucose,and glycated serum protein of glycolipid-related indices.The correlations were negative with body weight,Lee’s index,the above-mentioned body composition indexes,total cholesterol,triglycerides,LDL cholesterol,fasting glucose,and glycated serum protein,and positive with HDL cholesterol.3.Metabolomics-based analysis of the effects of different ratios of high-fat diets on rats and the intervention effect of Coix seedAfter metabolomic analysis,the common differential metabolites in the positive ion mode during the modeling phase were5-(8-Amino-1,2,3,4-tetrahydro-2-methyl-4-isoquinolinyl)-2-methoxyphenol,1-(9Z-Nonadecenoyl)-glycero-In the negative ion mode,the common differential metabolite in all groups was 13-Hydroxyoctadeca-9,12-dienoic acid.raw Coix seed and Coix seed together significantly back-regulated one differential metabolite: Leu-Arg-Asn-Arg.during the modeling period,compared with the normal group,the high-fat A total of 19 biomarkers in groups 1,2 and3 were mainly enriched in 4 metabolic pathways,mainly altered in linoleic acid,α-linolenic acid,arachidonic acid and glycerophospholipid metabolism pathways.During the administration period,7 biomarkers in the raw Coix seed group were mainly enriched to 4 metabolic pathways compared to the model group,and the main alterations were in amino sugar and nucleotide sugar metabolism,steroid biosynthesis metabolism,metabolism of xenobiotics by cytochrome P450,and purine metabolism related pathways.Conclusions:1.different ratios of high-fat diets caused the phenotype of phlegmatic obesity by altering the body mass of rats,affecting glucolipid metabolism,and increasing the overall water content,with the 5th month of modeling as the best state of this model.coix seeds can improve the dampness by improving the dyslipidemia,blood viscosity and liver lipid infiltration of rats in phlegm-damp obesity model.2.High-fat diets disrupt intestinal flora by changing the relative abundance and composition of intestinal flora in rats,and the degree of flora alteration is positively correlated with the fat content of feeds,among which high-fat group 3 has the greatest effect on intestinal flora.Both raw coix seed and French coix seed could play the role of dispelling moisture by adjusting the structure of flora through regulating the abundance and diversity of different flora.3.High-fat diets may affect the model rats through the metabolic pathways of glycerophospholipids,linoleic acid,α-linolenic acid,and arachidonic acid,resulting in phlegm and dampness obesity,and raw coix seed can exert its own expectorant effect by amino sugar and nucleotide sugar metabolism,steroid biosynthesis metabolism,metabolism of xenobiotics by cytochrome P450,and purine metabolism pathways in the high-fat diet model. |
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