| Objective: Due to the changes in the modern diet,obesity caused by overnutrition has become a worldwide epidemic,seriously endangering human health.Chinese medicine believes that nutritional obesity is caused by the thick and greasy diet,which leads to wet turbidity,and the dampness accumulates into a cream and forms obesity.It is adjuvant and aroma,and its representative drugs are musk and perrin.In order to study the "sweetening" effect of aromatized wet drug musk and peland,and its mechanism of action,this paper will study the lipid-lowering effect and mechanism of aromatized wet drug Musk and Perrin on nutritional obese rats and their relationship to bone marrow.The effects of adipogenic differentiation of mesenchymal stem cells were discussed.Method: 1.Establish a nutritional obese rat model by high fat diet induction 60 SPF SD rats weighing 120±10 g were fed for 1 week.According to the weight of the rats,the random function was divided into 10 groups in the normal group and 50 in the model group.The normal group was fed with normal feed,and the model was fed with imported high-fat diet to dynamically monitor the body weight and body length of the rats.After 4 weeks of feeding,the serum levels of triglyceride(TG),total cholesterol(CHOL),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C),and glucose(GLU)were measured.A nutritional obese rat model was established based on the body weight of the model rats,Lee’s index and serum TG content.2.Detection of Physiological and Biochemical Indexes in Rat Models of Nutritional Obesity by Musk and Perrin After the model was established successfully,the model was randomly divided into the model group,the musk high and low dose groups,and the Pelan high and low dose groups according to body weight and serum TG content.The low dose group of Musk and Perrin was administered at a dose of 3g/kg·d,and the high dose of Musk and Perrin was administered at a dose of 6g/kg·d(mixed volatile oil,according to the Chinese version of the 2015 edition of the Chinese Pharmacopoeia).Rat dose),drug intervention for 4 weeks.The normal group and the model group were given the same volume of physiological saline.During the drug intervention,the body weight and body length of the rats were monitored weekly.Drug intervention for 4 weeks,abdominal anesthesia,abdominal aorta blood sampling,detection of serum TG,CHOL,HDL-C,LDL-C,GLU content.3.Metabolomics research was used to study the changes of endogenous differential compounds in the lipid-lowering effects of musk and Pelan intervention in nutritional obese rats and the metabolic pathways in which they act.After 4 weeks of drug intervention,50 μL of serum samples from each group were extracted and placed in a 1.5 m L EP tube.150 μL of methanol was added and vortexed for 1 min.After completion,the serum samples were allowed to stand in a refrigerator at 4 ° C for 3 h and centrifuged for 10 min(15,000 rpm,4 ° C),the supernatant was extracted,nitrogen was blown,and 100 μL of methanol solution(methanol: water = 15:85)was used to dissolve the remaining residue.The sample was vortexed for 1 min,centrifuged for 15 min(18,000 rpm,4 ° C),and taken.The supernatant was analyzed by Agilent high performance liquid chromatography to find differential compounds and their metabolic pathways.4.Application of transcriptomics research to study the expression of differential genes in musk rats induced by musk and peline After 4 weeks of drug intervention,the rats were anesthetized for 24 hours,abdominal anesthesia,the liver of the animal was taken out,and the liver surface was cut off on the ice box.The internal tissues of the liver were quickly divided into several portions and placed in a cryotube containing RNA protection solution.Transfer to a-80 ° C refrigerator for storage.Total RNA was extracted by Tizol method and submitted to Huada Gene Company for testing and sequencing to find differentially expressed genes in each group.5.Effect of Musk and Perrin-containing Serum on Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells(1)Musk and Perrin were 500 g each,extracting the medicinal juice,adding volatile oil,and concentrating to 1g/m L;70 SPF SD rats,weighing 180±20g,were divided into normal group,musk and perill.After 7 days of gavage,the intragastric dose was 3g/kg·d,blood was taken from the abdominal aorta,and the serum was separated and stored at-20 °C.(2)Bone marrow mesenchymal stem cells were taken from SD rats and cultured for 4 generations for flow identification.(3)Configuration of inducer A: indomethacin,IBMX,insulin,dexamethasone(final concentration of 60 μmol/L,500 μmol/L,10 mg/L,1 μmol/L,respectively)in the medium;inducer Configuration of B: Insulin and rosiglitazone were added to the medium(final concentrations were 10 mg/L,1 μmol/L,respectively).(4)The experiment was divided into 7 experimental groups,normal group,model group,1.25%,2.5%,5%,10%,15% of the musk and perrin-containing drug groups,and inducer A was induced for 3 days,inducer B.Maintenance,for 21 days,oil red O staining,extraction,cell TG content detection.Result: 1.The model induced by high-fat diet was molded for 4 weeks.The weight gain of model animals was significantly higher than that of the normal group at the second week(P<0.01).The Lee’s index was also significantly higher than that of the normal group at the third week(P<0.01).).The body weight,Lee’s index,serum TG,GHOL,HDL-C,LDL-C and GLU levels in the 4th week were significantly higher than those in the normal group(P<0.01).The model of nutritional obesity was established successfully.2.Aromatized wet drug Pelan intervention in the nutritional obesity rat model,compared with the model group,the weight and Lee’s index of the Pelan high and low dose groups did not improve significantly(P> 0.05),Perlan low dose group rats The contents of TG,CHOL,LDL-C and GLU in serum decreased significantly(P<0.05),and the content of HDL-C increased significantly(P<0.01).In the high dose group,the TG content in serum decreased significantly(P<0.01).There was no significant change in the contents of GHOL,HDL-C,LDL-C and GLU(P>0.05).3.Based on metabolomics analysis,Pelan intervened in a rat model of nutritional obesity and found 12 potential biomarkers: lysine,arginine,tryptophan,valine,citrulline,pantothenic acid.Acrylic acid,acetaldehyde,dihydrosphingosine,corticosterone,pregnenolone,phytosphingosine,etc.These 12 different compounds mainly through tryptophan metabolism,arginine and proline Metabolic and sphingolipid metabolism play a role in three metabolic pathways.4.Based on transcriptomic analysis,it was found that Perrin intervention in the nutritional obesity rat model mainly through the mediation of PPAR signaling pathway,fatty acid metabolism insulin signaling pathway,glycerolipid metabolism,regulation of fat breakdown in fat cells,fatty acid biosynthesis of citric acid cycle Metabolic pathways such as TCA cycle down-regulate the expression of Fasn and Igfbp1 genes;up-regulate the expression of genes such as Acly and Socs2 to exert lipid-lowering effects.5.Aromatized wet musk intervention in the nutritional obesity rat model,compared with the model group,the body weight of the musk high-low dose group,Lee’s index and serum CHOL,HDL-C,LDL-C,GLU did not change significantly(P>0.05),but the serum TG content in the high-and low-dose groups was significantly decreased(P<0.01),and the high-dose musk group had a significant decrease in GHOL.6.Based on metabolomics analysis,it was found that Musk intervened in the rat model of nutritional obesity and found 10 endogenous potential biomarkers: ceramide,phytosphingosine,cytosine,hydrazine acrylic acid,pantothenic acid,methionine,tryptophan,tyramine,tyrosine,urea,etc.These 10 different compounds are mainly biosynthesized by phenylalanine,tyrosine and tryptophan,aminoacyl-t RNA biosynthesis,tyrosine metabolism The four metabolic pathways of sphingolipid metabolism play a role in lipid lowering.7.Based on transcriptome analysis,it was found that Musk intervention in the nutritional obesity rat model mainly down-regulated Fasn and Ppp1r3 b through 26 metabolic pathways including PPAR signaling pathway,fatty acid metabolism and insulin signaling pathway,and up-regulated the expression of Scd and Socs2 to exert lipid-lowering effects.8.In this experiment,oil red staining,extraction and cell TG content detection showed that 5%,10%,and 15% Perrin-containing serum groups significantly inhibited the adipogenic differentiation of BMSCs,and there was no significant concentration gradient between the groups.The 15% musk-containing serum group had a certain effect on reducing the adipogenic differentiation of BMSCs,but had no significant inhibitory effect on the TG content of cells.Conclusion: The aromatized wet drug Musk and Perrin intervene in the nutritional obesity caused by the accumulation of moist and turbid products and the accumulation of dampness,which has a certain lipid-lowering effect.The mechanism of action may be through intervention of metabolic pathways such as amino acid metabolism and lipid metabolism,down-regulating Fasn,up-regulating the expression of key genes of Socs2,inhibiting the synthesis of triglycerides,fatty acids and cholesterol,inhibiting the adipogenic differentiation of preadipocytes,and alleviating insulin resistance.The pathway exerts "sweetening" and lipid-lowering effects to interfere with the occurrence and development of nutritional obesity.In addition,the medicated serum of Musk and Perrin has certain effects on inhibiting the adipogenic differentiation of BMSCs.This experimental study shows that the lipid content in the body is one of the characteristics of "internal wetness" in traditional Chinese medicine. |
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