Effect Of Pu-erh Tea And Theabrownin In The Intervention Of Obesity In Rats And Its Mechanism Based On MicroRNA Regulation

Pu-er tea is rich in a variety of active ingredients,and the effect of lowering fat and losing weight is remarkable.TB is an important active ingredient of Pu-erh tea,but its potential anti-obesity mechanism has not been fully elucidated.As a novel mediator of nutritional regulation of biological functions,miRNAs play an important role in the regulation of obesity,and exploring the potential molecular mechanism of miRNA-mediated theabrownin in regulating obesity may help enrich the new targets of theabrownin regulating health.Therefore,in this study,high-fat diet induction was used to establish an obese rat model,while WE WE and TB intervention were carried out,16 S r RNA sequencing and metabolomics were used to study the changes of intestinal flora and endogenous metabolites in HFD rats,combined with miRNA sequencing methods and in vitro/in vivo experiments,to explore the correlation between the mechanism of action of WE and TB in regulating glycolipid metabolism disorders in high-fat diet mode and miRNA spectroscopy,and to find WE,TB may regulate key miRNAs.The results of the study are as follows:(1)Based on Curie point pyrolysis gas chromatography-mass spectrometry technology,the volatile cracking products of WE and TB at different pyrolysis temperatures were analyzed.At the thermal cracking temperature of 380 °C or 600 °C for WE and TB,the compounds with the highest relative content in the two sample pyrolysis products were caffeine,and the caffeine content in the WE lysate was higher than that in TB.(2)Untargeted metabolomics methods are based on untargeted metabolomics methods to reveal compositional differences between WE and TB.There were large metabolite differences between WE and TB,and a total of 58 differential metabolites were detected,mainly including 12 fatty acids,19 organic acids,3 alkaloids,4monomeric catechin derivatives,and 9 flavonoids,among which the content of alkaloids,flavonoids,monomeric catechins and their derivatives in WE was higher than that of TB,and TB contained more organic acids.(3)The effects of WE and TB on obesity prevention in rats on high-fat diet were clarified.The results showed that WE and TB could prevent rapid weight growth in high-fat dietary mode,reduce blood lipid blood glucose level and fat coefficient in rats,and both WE and TB could significantly reduce HOMA-IR with dose effects.At the same time,WE and TB can improve the inflammatory response induced by high lipids,reduce the levels of TNF-α,IL-6 and IL-1β in serum,and increase the content of Nrf2.In addition,WE and TB regulate the activities of key enzymes in lipid metabolism,enhance the activities of ATGL and HSL enzymes,significantly increase the activities of SOD,CAT,GSH-Px antioxidant enzymes,reduce the accumulation of MDA and alleviate hepatic steutosis,reduce the levels of TC and TG in the liver,and increase the FFA content.Overall,WE and TB have excellent effects in regulating obesity.(4)The results of 16 S rRNA sequencing showed that WE and TB could reverse the intestinal microbiota structure of rats with high-fat dietary patterns,restore the microbiota structure to normal levels,increase the Bacteroidetes/Firmicutes ratio in rats with HFD,and reduce the relative abundance of potentially harmful bacteria(Enterobacteriaceae,Helicobacter and Lachnoclostridium)with WE and TB.Increase the relative abundance of potentially beneficial bacteria(Bacteroides,Lactobacillus,and Bifidobacterium).This shows that WE and TB can target the regulation of gut microbial composition and the relative abundance of core microorganisms to alleviate obesity.(5)Non-targeted metabolomics analysis of metabolites of rat cecal contents and screening for differential metabolites related to WE and TB lipid-lowering effects.WE down-regulated 36 differential metabolites such as corticosterone,glusyl alcohol,and ursodeoxycholic acid;Upregulated nine differential metabolites,including hydrogenated cinnamic acid,(R)-3-hydroxybutyric acid and L-phenylalanine;Compared with the HFD group,TB upregulated 13 differential metabolites such as mannan heptitol,glycerophosphocholine and L-histidine,and reduced 8 differential metabolites such as alfalfalic acid,2-methylpyrrole,and mannitol.The alleviation of obesity by WE and TB is closely related to phenylalanine metabolism,glycerophospholipid metabolism,steroid hormone biosynthesis,arachidonic acid metabolism and caffeine metabolism.(6)The effect of WE and TB on liver miRNA expression in rats under high-fat diet was explored by miRNA sequencing technology,and miRNAs related to WE and TB lipid-lowering effects were screened.Compared with the HFD group,the expression levels of miR-125b-5p,miR-223-3p＿R+2,miR-148b-3p and miR-1247-5p were significantly downregulated by WE and TB,and there was no significant difference between the expression levels of the normal group.Differential miRNA target genes were significantly enriched in signaling pathways such as MAPK,P13 KAKT,AMPK,AND FOXO,AND DIFFERENTIAL MIRNAs were significantly correlated with obesity-related indexes(P < 0.05).It is speculated that differential miRNAs may be potential regulators of HFD-induced obesity by WE and TB.(7)It was verified that WE and TB regulated obesity in rats dependent on miRNA expression,and WE and TB significantly upregulated the expression levels of AKT,PGC-1a and G6 pc genes in the PI3K-AKT-FOXO signaling pathway,significantly downregulated the expression levels of FOXO,PGC-1a and G6 Pase,and miRNA overexpression inhibited PI3K-AKT-FOXO signaling.This paper showed that WE and TB regulated glycolipid metabolism by inhibiting abnormally expressed miRNAs and activating the PI3K-AKT-FOXO signaling pathway.(8)The specific molecular mechanism of differential miRNA in regulating glycolipid metabolism was revealed,and the target of miRNA was clarified.P13KAKT-FOXO signaling pathway signaling molecules,including Grb2,pik3r3,Pik3 ca and Gng10 genes were screened as candidate target genes.WE and TB significantly upregulated the expression level of miRNA target genes,while miRNA overexpression significantly reduced the expression level of target genes.In addition,co-transfection of the target gene wild-type reporter vector with miRNA led to a significant decrease in luciferase activity,and the results determined the site of action of miRNA.This study showed that WE and TB effectively regulate glycolipid metabolism,improve non-alcoholic fatty liver disease,increase insulin sensitivity,and inhibit the preventive effects of oxidative stress and inflammatory response.The improvement of the above phenotypes by WE and TB was achieved by regulating changes in intestinal flora,metabolite composition,and the miRNA-PI3K-AKT-FOXO axis.